Thermostable DNA ligase mutants

ABSTRACT

Ligase detection reaction is utilized to distinguish minority template in the presence of an excess of normal template with a thermostable ligase. This process can be carried out with a mutant ligase, thermostable ligase, or a modified oligonucleotide probe. This procedure is particularly useful for the detection of cancer-associated mutations. It has the advantage of providing a quantitative measure of the amount or ratio of minority template.

This application is a division of U.S. patent application Ser. No.08/891,292, filed Jul. 10, 1997, now U.S. Pat. No. 6,312,892, and claimsthe benefit of U.S. Provisional Patent Application Serial No.60/022,535, filed Jul. 19, 1996.

This invention was developed with government funding under NationalInstitutes of Health Grant No. GM41337-06. The U.S. Government mayretain certain rights.

FIELD OF THE INVENTION

The present invention relates to the high fidelity detection of nucleicacid sequence differences using ligase detection reaction (“LDR”). Oneaspect of the present invention involves use of a ligase detectionreaction to distinguish minority template in the presence of an excessof normal template with a thermostable ligase. Another aspect of thepresent invention relates to the use of a mutant ligase to carry out aligase detection reaction. A third aspect of the present inventioninvolves use of a modified oligonucleotide probe to carry out a ligasedetection reaction.

BACKGROUND OF THE INVENTION

Multiplex Detection

Large-scale multiplex analysis of highly polymorphic loci is needed forpractical identification of individuals, e.g., for paternity testing andin forensic science (Reynolds et al., Anal. Chem., 63:2-15 (1991)), fororgan-transplant donor-recipient matching (Buyse et al., TissueAntigens, 41:1-14 (1993) and Gyllensten et al., PCR Meth. Appl, 1:91-98(1991)), for genetic disease diagnosis, prognosis, and pre-natalcounseling (Chamberlain et al., Nucleic Acids Res., 16:11141-11156(1988) and L. C. Tsui, Human Mutat., 1:197-203 (1992)), and the study ofoncogenic mutations (Holistein et al., Science, 253:49-53 (1991)). Inaddition, the cost-effectiveness of infectious disease diagnosis bynucleic acid analysis varies directly with the multiplex scale in paneltesting. Many of these applications depend on the discrimination ofsingle-base differences at a multiplicity of sometimes closely spacedloci.

A variety of DNA hybridization techniques are available for detectingthe presence of one or more selected polynucleotide sequences in asample containing a large number of sequence regions. In a simplemethod, which relies on fragment capture and labeling, a fragmentcontaining a selected sequence is captured by hybridization to animmobilized probe. The captured fragment can be labeled by hybridizationto a second probe which contains a detectable reporter moiety.

Another widely used method is Southern blotting. In this method, amixture of DNA fragments in a sample is fractionated by gelelectrophoresis, then fixed on a nitrocellulose filter. By reacting thefilter with one or more labeled probes under hybridization conditions,the presence of bands containing the probe sequences can be identified.The method is especially useful for identifying fragments in arestriction-enzyme DNA digest which contains a given probe sequence andfor analyzing restriction-fragment length polymorphisms (“RFLPs”).

Another approach to detecting the presence of a given sequence orsequences in a polynucleotide sample involves selective amplification ofthe sequence(s) by polymerase chain reaction. U.S. Pat. No. 4,683,202 toMullis, et al. and R. K. Saiki, et al., Science 230:1350 (1985). In thismethod, primers complementary to opposite end portions of the selectedsequence(s) are used to promote, in conjunction with thermal cycling,successive rounds of primer-initiated replication. The amplifiedsequence(s) may be readily identified by a variety of techniques. Thisapproach is particularly useful for detecting the presence of low-copysequences in a polynucleotide-containing sample, e.g., for detectingpathogen sequences in a body-fluid sample.

More recently, methods of identifying known target sequences by probeligation methods have been reported. U.S. Pat. No. 4,883,750 to N. M.Whiteley, et al., D. Y. Wu, et al., Genomics 4:560 (1989), U. Landegren,et al., Science 241:1077 (1988), and E. Winn-Deen, et al., Clin. Chem.37:1522 (1991). In one approach, known as oligonucleotide ligation assay(“OLA”), two probes or probe elements which span a target region ofinterest are hybridized to the target region. Where the probe elementsbasepair with adjacent target bases, the confronting ends of the probeelements can be joined by ligation, e.g., by treatment with ligase. Theligated probe element is then assayed, indicating the presence of thetarget sequence.

In a modification of this approach, the ligated probe elements act as atemplate for a pair of complementary probe elements. With continuedcycles of denaturation, hybridization, and ligation in the presence ofpairs of probe elements, the target sequence is amplified linearly,allowing very small amounts of target sequence to be detected and/oramplified. This approach is referred to as ligase detection reaction.When two complementary pairs of probe elements are utilized, the processis referred to as the ligase chain reaction which achieves exponentialamplification of target sequences. F. Barany, “Genetic Disease Detectionand DNA Amplification Using Cloned Thermostable Ligase,” Proc. Nat'lAcad. Sci. USA, 88:189-93 (1991) and F. Barany, “The Ligase ChainReaction (LCR) in a PCR World,” PCR Methods and Applications, 1:5-16(1991).

Another scheme for multiplex detection of nucleic acid sequencedifferences is disclosed in U.S. Pat. No. 5,470,705 to Grossman et. al.where sequence-specific probes, having a detectable label and adistinctive ratio of charge/translational frictional drag, can behybridized to a target and ligated together. This technique was used inGrossman, et. al., “High-density Multiplex Detection of Nucleic AcidSequences: Oligonucleotide Ligation Assay and Sequence-codedSeparation,” Nucl. Acids Res. 22(21):4527-34 (1994) for the large scalemultiplex analysis of the cystic fibrosis transmembrane regulator gene.

Jou, et. al., “Deletion Detection in Dystrophia Gene by Multiplex GapLigase Chain Reaction and Immunochromatographic Strip Technology,” HumanMutation 5:86-93 (1995) relates to the use of a so called “gap ligasechain reaction” process to amplify simultaneously selected regions ofmultiple exons with the amplified products being read on animmunochromatographic strip having antibodies specific to the differenthaptens on the probes for each exon.

There is a growing need (e.g., in the field of genetic screening) formethods useful in detecting the presence or absence of each of a largenumber of sequences in a target polynucleotide. For example, as many as400 different mutations have been associated with cystic fibrosis. Inscreening for genetic predisposition to this disease, it is optimal totest all of the possible different gene sequence mutations in thesubject's genomic DNA, in order to make a positive identification of“cystic fibrosis”. It would be ideal to test for the presence or absenceof all of the possible mutation sites in a single assay. However, theprior-art methods described above are not readily adaptable for use indetecting multiple selected sequences in a convenient, automatedsingle-assay format.

Solid-phase hybridization assays require multiple liquid-handling steps,and some incubation and wash temperatures must be carefully controlledto keep the stringency needed for single-nucleotide mismatchdiscrimination. Multiplexing of this approach has proven difficult asoptimal hybridization conditions vary greatly among probe sequences.

Developing a multiplex PCR process that yields equivalent amounts ofeach PCR product can be difficult and laborious. This is due tovariations in the annealing rates of the primers in the reaction as wellas varying polymerase extension rates for each sequence at a given Mg²⁺concentration. Typically, primer, Mg²⁺, and salt concentrations, alongwith annealing temperatures are adjusted in an effort to balance primerannealing rates and polymerase extension rates in the reaction.Unfortunately, as each new primer set is added to the reaction, thenumber of potential amplicons and primer dimers which could formincreases exponentially. Thus, with each added primer set, it becomesincreasingly more difficult and time consuming to work out conditionsthat yield relatively equal amounts of each of the correct products.

Allele-specific PCR products generally have the same size, and an assayresult is scored by the presence or absence of the product band(s) inthe gel lane associated with each reaction tube. Gibbs et al., NucleicAcids Res., 17:2437-48 (1989). This approach requires splitting the testsample among multiple reaction tubes with different primer combinations,multiplying assay cost. In PCR, discrimination of alleles can beachieved by attaching different fluorescent dyes to competing allelicprimers in a single reaction tube (F. F. Chehab, et al., Proc. Natl.Acad. Sci. USA, 86:9178-9182 (1989)), but this route to multiplexanalysis is limited in scale by the relatively few dyes which can bespectrally resolved in an economical manner with existinginstrumentation and dye chemistry. The incorporation of bases modifiedwith bulky side chains can be used to differentiate allelic PCR productsby their electrophoretic mobility, but this method is limited by thesuccessful incorporation of these modified bases by polymerase, and bythe ability of electrophoresis to resolve relatively large PCR productswhich differ in size by only one of these groups. Livak et al., NucleicAcids Res., 20:4831-4837 (1989). Each PCR product is used to look foronly a single mutation, making multiplexing difficult.

Ligation of allele-specific probes generally has used solid-phasecapture (U. Landegren et al., Science, 241:1077-1080 (1988); Nickersonet al., Proc. Natl. Acad. Sci. USA, 87:8923-8927 (1990)) orsize-dependent separation (D. Y. Wu, et al., Genomics, 4:560-569 (1989)and F. Barany, Proc. Natl. Acad. Sci., 88:189-193 (1991)) to resolve theallelic signals, the latter method being limited in multiplex scale bythe narrow size range of ligation probes. Further, in a multiplexformat, the ligase detection reaction alone cannot make enough productto detect and quantify small amounts of target sequences. The gap ligasechain reaction process requires an additional step—polymerase extension.The use of probes with distinctive ratios of charge/translationalfrictional drag for a more complex multiplex process will either requirelonger electrophoresis times or the use of an alternate form ofdetection.

The need thus remains for a rapid single assay format to detect thepresence or absence of multiple selected sequences in a polynucleotidesample when those sequences are in low abundance. Such detection isrequired when cancer-associated mutations are present in an excess ofnormal cells.

DNA Ligase

DNA ligases catalyze the formation of phosphodiester bonds atsingle-stranded breaks (nicks) in double-stranded DNA, and are requiredin DNA replication, repair, and recombination. The general mechanism ofligation reactions involves three reversible steps, as shown below forNAD⁺-dependent ligases. In this example, the nicked DNA substrate isformed by annealing two short oligonucleotides (oligo A and B) to alonger complementary oligonucleotide. First, a covalently adenylatedenzyme intermediate is formed by transfer of the adenylate group of NAD⁺to the e-NH₂ group of lysine in the enzyme. Second, the adenylate moietyis transferred from the enzyme to the 5′-terminal phosphate on oligo B.Finally, a phosphodiester bond is formed by a nucleophilic attack of the3′-hydroxyl terminus of oligo A on the activated 5′-phosphoryl group ofoligo B (Gumport, R. I., et al., Proc. Natl. Acad. Sci. USA, 68:2559-63(1971); Modrich, P., et al., J. Biol. Chem., 248:7495-7501 (1973);Modrich, P., et al., J. Biol. Chem., 248:7502-11 (1973); Weiss, B., etal., J. Biol. Chem., 242:4270-72 (1967); Weiss, B., et al., J. Biol.Chem., 243:4556-63 (1968); Becker, A., et al., Proc. Natl. Acad. Sci.USA, 58:1996-2003 (1967); Yudelevich, A., et al., Proc. Natl. Acad. Sci.USA, 61:1129-36 (1968); Zimmerman, S. B., et al., Proc. Natl. Acad. Sci.USA, 57:1841-48 (1967); Zimmerman, S. B., et al., J. Biol. Chem.,244:4689-95 (1969); and Lehman, I. R., Science, 186:790-97 (1974)).

(i) E-(lys)-NH₂+AMP˜PRN⁺⇄E-(lys)-NH₂ ⁺˜AMP+NMN

(ii) E-(lys)-NH₂ ⁺˜AMP+5′ P-Oligo B⇄AMP˜P-Oligo B+E-(lys)-NH₂

(iii) Oligo A-3′OH+AMP˜P-Oligo B⇄Oligo A-P-Oligo B+AMP

Within the last decade, genes encoding ATP-dependent DNA ligases havebeen cloned and sequenced from bacteriophages T3, T4, and T7 (Dunn, J.J., et al., J. Mol. Biol., 148:303-30 (1981); Armstrong, J., et al.,Nucleic Acids Res., 11:7145-56 (1983); and Schmitt, M. P., et al., J.Mol. Biol., 193:479-95 (1987)), African swine fever virus (Hammond, J.M., et al., Nucleic Acids Res., 20:2667-71 (1992)), Vaccinia virus(Smith, G. L., et al., Nucleic Acids Res., 17:9051-62 (1989)), Shopefibroma virus (Parks, R. J., et al., Virology, 202:642-50 (1994)), anextremely thermophilic archaeon Desulfurolobus ambivalens (Kletzin, A.,Nucleic Acids Res., 20:5389-96 (1992)), S. cerevisiae (CDC9gene)(Barker, D. G., et al., Mol. Gen. Genet., 200:458-62 (1985)); S.pombe (cdc17⁺) (Barker, D. G., et al., Eur. J. Biochem., 162:659-67(1988)), Xiphophorus (Walter, R. B., et al., Mol. Biol. Evol.,10:1227-38 (1993)); mouse fibroblast (Savini, E., et al., Gene,144:253-57 (1994)); and Homo sapiens (human DNA ligase I, III, and IV)(Barnes, D. E., et al., Proc. Natl. Acad. Sci. USA, 87:6679-83 (1990);Chen, J., et al., Molec. and Cell. Biology, 15:5412-22 (1995); and Wei,Y. F., et al., Molec. & Cell. Biology, 15:3206-16 (1995)). In addition,five NAD⁺-dependent bacterial DNA ligases have also been cloned: E. coli(Ishino, Y., et al., Mol. Gen. Genet., 204:1-7 (1986)), Zymomonasmobilis (Shark, K. B., et al., FEMS Microbiol. Lett., 96:19-26 (1992)),Thermus thermophilus (Barany, F., et al., Gene, 109:1-11 (1991) andLauer, G., et al., J. Bacteriol., 173:5047-53 (1991)), Rhodothermusmarinus (Thorbjarnardottir, S. H., et al., Gene, 161:1-6 (1995)), andThermus scotoductus (Jonsson, Z. O., et al., Gene, 151:177-80 (1994)).ATP-dependent DNA ligases, as well as mammalian DNA ligases I and IIcontain a conserved active site motif, K(Y/A)DGXR, which includes thelysine residue that becomes adenylated (Tomkinson, A. E., et al, Proc.Natl. Acad. Sci. USA, 88:400-04 (1991) and Wang, Y. C., et al., J. Biol.Chem., 269:31923-28 (1994)). NAD⁺-dependent bacterial DNA ligasescontain a similar active site motif, KXDG, whose importance is confirmedin this work.

In vitro experiments using plasmid or synthetic oligonucleotidesubstrates reveal that T4 DNA ligase exhibits a relaxed specificity;sealing nicks with 3′- or 5′-AP sites (apurinic or apyrimidinic)(Goffin, C., et al., Nucleic Acids Res., 15(21):8755-71 (1987)),one-nucleotide gaps (Goffin, C., et al., Nucleic Acids Res.,15(21):8755-71 (1987)), 3′- and 5′-A-A or T-T mismatches (Wu, D. Y., etal., Gene, 76:245-54 (1989)), 5′-G-T mismatches (Harada, K., et al.,Nucleic Acids Res., 21(10):2287-91 (1993)), 3′-C-A, C-T, T-G, T-T, T-C,A-C, G-G, or G-T mismatches (Landegren, U., et al., Science, 241:1077-80(1988)). The apparent fidelity of T4 DNA ligase may be improved in thepresence of spermidine, high salt, and very low ligase concentration,where only T-G or G-T mismatch ligations were detected (Wu, D. Y., etal., Gene, 76:245-54 (1989) and Landegren, U., et al., Science,241:1077-80 (1988)). DNA ligase from Saccharomyces cerevisiaediscriminates 3′-hydroxyl and 5′-phosphate termini separated by aone-nucleotide gap and 3′-A-G or T-G mismatches, however 5′-A-C, T-C,C-A, or G-A mismatches had very little effect on ligation efficiency(Tomkinson, A. E., et al., Biochemistry, 31:11762-71 (1992)). MammalianDNA ligases I and III show different efficiencies in ligating 3′ C-T,G-T, and T-G mismatches (Husain, I., et al., J. Biol. Chem., 270:9638-90(1995)). The Vaccinia DNA virus efficiently discriminates againstone-nucleotide, two-nucleotide gaps and 3′-G-A, A-A, G-G, or A-G(purine-purine) mismatches, but easily seals 5′-C-T, G-T, T-T, A-C, T-C,C-C, G-G, T-G, or A-G mismatches as well as 3′-C-A, C-T, G-T, T-T, orT-G mismatches (Shuman, S., Biochem., 34:16138-47 (1995)).

The thermostable Thermus thermophilus DNA ligase (Tth DNA ligase) hasbeen cloned and used in the ligase chain reaction (LCR) and ligasedetection reaction (LDR) for detecting infectious agents and geneticdiseases (Barany, F., Proc. Natl. Acad. Sci. USA, 88:189-93 (1991); Day,D., et al., Genomics, 29:152-62 (1995); Eggerding, F. A., PCR Methodsand Applications, 4:337-45 (1995); Eggerding, F. A., et al., HumanMutation, 5:153-65 (1995); Feero, W., et al., Neurology, 43:668-73(1993); Frenkel, L. M., et al., J. Clin. Micro., 33(2):342-47 (1995);Grossman, P. D., et al., Nucleic Acids Res., 22:4527-34 (1994);Iovannisci, D. M., et al., Mol. Cell. Probes, 7:35-43 (1993); Prchal, J.T., et al., Blood, 81:269-71 (1993); Ruiz-Opaz, N., et al.,Hypertension, 24:260-70 (1994); Wiedmann, M., et al., Appl. Environ.Microbiol., 58:3443-47 (1992); Wiedmann, M., et al., Appl EnvironMicrobiol, 59(8):2743-5 (1993); Winn-Deen, E., et al., Amer. J. HumanGenetics, 53:1512 (1993); Winn-Deen, E. S., et al., Clin. Chem., 40:1092(1994); and Zebala, J., et al., “Detection of Leber's Hereditary OpticNeuropathy by nonradioactive-LCR. PCR Strategies,” (Innis, M. A.,Gelfand, D. H., and Sninsky, J. J., Eds.), Academic Press, San Diego(1996)). The success of these and future disease detection assays, suchas identifying tumor associated mutations in an excess of normal DNA,depend on the exquisite fidelity of Tth DNA ligase.

Cancer Detection

As the second leading cause of death in this country, almost 600,000people will die from cancer per year making cancer one of the mostalarming of all medical diagnosis. Lifetime risks for developinginvasive cancers in men and women are 50 percent and 33 percent,respectively. Expectations are that more than 1.2 million new cases ofcancer will be diagnosed in the United States in 1995. Healthcareexpenses for cancer in 1994 were approximately $104 billion. However,the full impact of cancer on families and society is not measured onlyby the amount of money spent on its diagnosis and treatment. Asignificant number of people are stricken with cancer in their mostproductive years. Cancers accounted for 18 percent of premature deathsin 1985 and in 1991 more than 9,200 women in the U.S. died from breastcancer before the age of 55. For colorectal and breast cancers,estimates are that nearly 140,000 and 183,000 new diagnoses,respectively, are predicted for 1995.

Currently, diagnosis of cancer is based on histological evaluation oftumor tissue by a pathologist. After a cancer is diagnosed, treatment isdetermined primarily by the extent or stage of the tumor. Tumor stage isdefined by clinical, radiological, and laboratory methods. Standardizedclassification systems for the staging of tumors have been developed toclearly convey clinical information about cancer patients. Stagingprovides important prognostic information and forms the basis ofclinical studies which allow the testing of new treatment strategies. Astaging system was developed (TNM staging system), which classifiestumors according to the size of the primary tumor, the number ofregional lymph nodes in which cancer is found, and the presence orabsence of metastases to other parts of the body. Smaller cancers withno affected lymph nodes and no distant metastases are considered earlystage cancers, which are often amenable to cure through surgicalresection. A common measure of prognosis is the 5-year survival rate,the proportion of patients alive five years after the diagnosis of acancer at a given stage. While 5-year survival rates for many cancershave improved over the last few decades, the fact that some early stagecancers recur within five years or later has led researchers to exploreother additional prognostic markers including histological grade,cytometry results, hormone receptor status, and many other tumormarkers. Most recently, investigators have explored the use of molecularalterations in cancers as markers of prognosis.

Genetic alterations found in cancers, such as point mutations and smalldeletions mentioned above, can act as markers of malignant cells.

Detection of Minority Nucleic Acid Sequences

A number of procedures have been disclosed to detect cancer using PCR.Sidransky, et. al., “Identification of ras Oncogene Mutations in theStool of Patients with Curable Colorectal Tumors,” Science 256: 102-05(1992) detects colon cancer by identification of K-ras mutations. Thisinvolves a PCR amplification of total DNA, cloning into a phage vector,plating out the phage, repeated probing with individual oligonucleotidesspecific to several different K-ras mutations, and counting thepercentage of positive plaques on a given plate. This is a technicallydifficult procedure which takes three days to complete, whereby theratio of mutant to wild-type DNA in the stool sample is determined.Brennan, et. al., “Molecular Assessment of Histopathological Staging inSquamous-Cell Carcinoma of the Head and Neck,” N. Engl. J. Med. 332(7):429-35 (1995) finds p53 mutations by sequencing. This specific mutationis then probed for in margin tissue using PCR amplification of totalDNA, cloning into a phage vector, plating out the phage, probing with anindividual oligonucleotide specific to the mutation found by sequencing,and counting the percentage of positive plaques on a given plate.Berthelemy, et. al., “Brief Communications—Identification of K-rasMutations in Pancreatic Juice in the Early Diagnosis of PancreaticCancer,” Ann. Int. Med. 123(3): 188-91 (1995) uses a PCR/restrictionenzyme process to detect K-ras mutations in pancreatic secretions. Thistechnique is deficient, however, in that mutations are not quantified.Similarly, Tada, et. al., “Detection of ras Gene Mutations in PancreaticJuice and Peripheral Blood of Patients with Pancreatic Adenocarcinoma,”Cancer Res. 53: 2472-74 (1993) and Tada, et. al., “Clinical Applicationof ras Gene Mutation for Diagnosis of Pancreatic Adenocarcinoma,”Gastroent. 100: 233-38 (1991) subject such samples to allele-specificPCR to detect pancreatic cancer. This has the disadvantages of providingfalse positives due to polymerase extension off normal template,requiring electrophoretical separation of products to distinguish fromprimer dimers, being unable to multiplex closely-clustered sites due tointerference of overlapping primers, being unable to detect single baseor small insertions and deletions in small repeat sequences, and notbeing ideally suitable for quantification of mutant DNA in a highbackground of normal DNA. Hayashi, et. al., “Genetic DetectionIdentifies Occult Lymph Node Metastases Undetectable by theHistopathological Method,” Cancer Res. 54: 3853-56 (1994) uses anallele-specific PCR technique to find K-ras or p53 mutations to identifyoccult lymph node metastases in colon cancers. A sensitivity of onetumor cell in one thousand of normal cells is claimed; however,obtaining quantitative values requires laborious cloning, plating, andprobing procedures. In Mitsudomi, et. al., “Mutations of ras GenesDistinguish a Subset of Non-small-cell Lung Cancer Cell Lines fromSmall-cell Lung Cancer Cell Lines,” Oncogene 6: 1353-62 (1991), humanlung cancer cell lines are screened for point mutations of the K-, H-,and N-ras genes using restriction fragment length polymorphisms createdthrough mismatched primers during PCR amplification of genomic DNA. Thedisadvantages of such primer-mediated RFLP include the requirement ofelectrophoretical separation to distinguish mutant from normal DNA,limited applicability to sites that may be converted into a restrictionsite, the requirement for additional analysis to determine the nature ofthe mutation, and the difficulty in quantifying mutant DNA in a highbackground of normal DNA. Further, these procedures tend to be laboriousand inaccurate.

Coupled PCR/ligation processes have been used for detection of minoritynucleotide sequences in the presence of majority nucleotide sequences. APCR/LDR process is used in Frenkel, “Specific, Sensitive, and RapidAssay for Human Immunodeficiency Virus Type 1 pol Mutations Associatedwith Resistance to Zidovudine and Didanosine,” J. Clin. Microbiol.33(2): 342-47 (1995) to detect HIV mutants. This assay, however, cannotbe used for multiplex detection. See also Abravaya, et. al., “Detectionof Point Mutations With a Modified Ligase Chain (Gap-LCR),” Nucl. AcidsRes. 23(4): 675-82 (1995) and Balles, et. al., “Facilitated Isolation ofRare Recombinants by Ligase Chain Reaction: Selection for IntragenicCrossover Events in the Drosophila optomotor-blind Gene,” Molec. Gen.Genet. 245: 734-40 (1994).

Colorectal lesions have been detected by a process involving PCRamplification followed by an oligonucleotide ligation assay. See Jen,et. al., “Molecular Determinants of Dysplasia in Colorectal Lesions,”Cancer Res. 54: 5523-26 (1994) and Redston, et. al., “Common Occurrenceof APC and K-ras Gene Mutations in the Spectrum of Colitis-AssociatedNeoplasias,” Gastroenter. 108: 383-92 (1995). This process was developedas an advance over Powell, et. al., “Molecular Diagnosis of FamilialAdenomatous Polyposis,” N. Engl. J. Med. 329(27): 1982-87 (1993). Thesetechniques tend to be limited and difficult to carry out.

Other procedures have been developed to detect minority nucleotidesequences. Lu, et. al., “Quanititative Aspects of the Mutant Analysis byPCR and Restriction Enzyme Cleavage (MAPREC)” PCR Methods and Appl. 3:176-80 (1993) detects virus revertants by PCR and restriction enzymecleavage. The disadvantages of MAPREC include the requirement forelectrophoretical separation to distinguish mutant from normal DNA,limited applicability to sites that may be converted into a restrictionsite, the requirement for additional analysis to determine the nature ofthe mutation, and difficulty in quantifying mutant DNA in a highbackground of normal DNA. In Kuppuswamy, et. al., “Single NucleotidePrimer Extension to Detect Genetic Diseases: Experimental Application toHemophilia G (Factor IX) and Cystic Fibrosis Genes,” Proc. Natl. Acad.Sci. USA 88: 114347 (1991), a PCR process is carried out using 2reaction mixtures for each fragment to be amplified with one mixturecontaining a primer and a labeled nucleotide corresponding to the normalcoding sequence, while the other mixture contains a primer and a labelednucleotide corresponding to the mutant sequence. The disadvantages ofsuch mini sequencing (i.e. SNuPe) are that the mutations must be known,it is not possible to multiplex closely clustered sites. due tointerference of overlapping primers, it is not possible to detect singlebase or small insertions and deletions in small repeat sequences, andfour separate reactions are required. A mutagenically separated PCRprocess is disclosed in Rust, et. al., “Mutagenically Separated PCR(MS-PCR): a Highly Specific One Step Ptocedure for easy MutationDetection” Nucl. Acids Res. 21(16): 3623-29 (1993) to distinguish normaland mutant alleles, using different length allele-specific primers. Thedisadvantages of MS-PCR include possibly providing false positives dueto polymerase extension off normal template, requiring electrophoreticalseparation of products to distinguish from primer dimers, the inabilityto multiplex closely-clustered sites due to interference of overlappingprimers, the inability to detect single base or small insertions anddeletions in small repeat sequences, and not being ideally suited forquantification of mutant DNA in high background of normal DNA. InSuzuki, et. al., “Detection of ras Gene Mutations in Human Lung Cancersby Single-Strand Conformation Polymorphism Analysis of Polymerase ChainReaction Products,” Oncogene 5: 103743 (1990), mutations are detected ina process having a PCR phase followed by phase involving single strandconformation polymorphism (“SSCP”) of the amplified DNA fragments. Thedisadvantages of SSCP include the requirement for electrophoreticalseparation to distinghish mutant conformer from normal conformer, thefailure to detect 30% of possible mutations, the requirement foradditional analysis to determine the nature of the mutation, and theinability to distinguish mutant from silent polymorphisms.

Despite the existence of techniques for detecting minority nucleotidesequences in the presence of majority sequences, the need remains forimproved procedures of doing so. It is particularly desirable to developsuch techniques where minority nucleotide sequences can be quantified.

SUMMARY OF THE INVENTION

One aspect of the present invention relates to a method for detecting ina sample one or more minority target nucleotide sequences which differfrom one or more majority target nucleotide sequences by one or moresingle-base changes, insertions, deletions, or translocations, whereinthe minority target nucleotide sequences are present in the sample inlesser amounts than the majority nucleotide sequences.

One or more oligonucleotide probe sets are provided for use inconjunction with this method. Each set includes (a) a firstoligonucleotide probe having a target-specific portion and (b) a secondoligonucleotide probe having a target-specific portion. Theoligonucleotide probes in a particular set are suitable for ligationtogether when hybridized adjacent to one another on a correspondingtarget nucleotide sequence, but have a mismatch which interferes withsuch ligation when hybridized to any other nucleotide sequence presentin the sample.

The sample, the one or more oligonucleotide probe sets, and a ligase areblended to form a ligase detection reaction mixture. The ligasedetection reaction mixture is subjected to one or more ligase detectionreaction cycles comprising a denaturation treatment and a hybridizationtreatment. In the denaturation treatment, any hybridizedoligonucleotides are separated from the target nucleotide sequences. Thehybridization treatment involves hybridizing the oligonucleotide probesets at adjacent positions in a base-specific manner to the respectivetarget nucleotide sequences, if present in the sample. The hybridizedoligonucleotide probes from each set ligate to one another to form aligation product sequence containing the target-specific portionsconnected together. The ligation product sequence for each set isdistinguishable from other nucleic acids in the ligase detectionreaction mixture. The oligonucleotide probe sets may hybridize toadjacent sequences in the sample other than the respective targetnucleotide sequences but do not ligate together due to the presence ofone or more mismatches. When hydridized oligonucleotide probes do notligate, they individually separate during the denaturation treatment.

After the ligase detection reaction mixture is subjected to one or moreligase detection reaction cycles, ligation product sequences aredetected. As a result, the presence of the minority target nucleotidesequence in the sample can be identified.

The second aspect of the present invention also relates to a method foridentifying one or more of a plurality of sequences differing by one ormore single-base changes, insertions, deletions, or translocations in aplurality of target nucleotide sequences. As noted above, a sample andone or more oligonucleotide probe sets are blended with a ligase to forma ligase detection reaction mixture. The ligase detection reactionmixture is subjected to one or more ligase detection reaction cycles,and the presence of ligation product sequences is detected. Here,however, a thermostable mutant ligase is utilized. This ligase ischaracterized by a fidelity ratio which is defined as the initial rateconstant for ligating the first and second oligonucleotide probeshybridized to a target nucleotide sequence with a perfect match at theligation junction between the target nucleotide sequence and theoligonucleotide probe having its 3′ end at the ligation junction to theinitial rate constant for ligating the first and second oligonucleotideprobes hybridized to a target with a mismatch at the ligation junctionbetween the target nucleotide sequence and the oligonucleotide probehaving its 3′ end at the ligation junction. The fidelity ratio for thethermostable mutant ligase is greater than the fidelity ratio forwild-type ligase.

The third aspect of the present invention also relates to a method foridentifying one or more of a plurality of sequences differing by one ormore single-base changes, insertions, deletions, or translocations in aplurality of target nucleotide sequences. As noted above, a sample andone or more oligonucleotide probe sets are blended with a ligase to forma ligase detection reaction mixture. The ligase detection reactionmixture is subjected to one or more ligase detection reaction cycles,and the presence of ligation product sequences is then detected. Here,however, with regard to the oligonucleotide probe sets, theoligonucleotide probe which has its 3′ end at the junction whereligation occurs has a modification. This modification differentiallyalters the ligation rate when the first and second oligonucleotideprobes hybridize to a minority target nucleotide sequence in the samplewith a perfect match at the ligation junction between the minoritytarget nucleotide sequence and the oligonucleotide probe having its 3′end at the ligation junction compared to the ligation rate when thefirst and second oligonucleotide probes hybridize to the sample'smajority target nucleotide sequence with a mismatch at the ligationjunction between the majority target nucleotide sequence and thenucleotide probe having its 3′ end at the ligation junction. Ligationwith the modified oligonucleotide probe has a signal-to-noise ratio, ofthe ligation product sequence amounts for the minority and majoritytarget nucleotide sequences to the amount of ligation product sequencesproduced from the same amount of majority target sequence alone, whichis greater than the signal-to-noise ratio for ligation using anoligonucleotide probe lacking the modification.

In developing a procedure for detection of cancer-associated mutationsor the presence of a minority target nucleotide sequence, it isnecessary for the procedure to be capable of diagnosing cancer at anearly stage. This requires that at least one clonal mutation beidentified and accurately quantified from clinical samples. An idealtest of this type would rapidly screen up to hundreds of commonmutations in multiple genes. It must accurately quantify less than 1%mutant DNA in the presence of normal DNA and correctly distinguish manyclosely clustered mutations in a multiplex format. For point mutationsgenerally and, in small repeat sequences particularly, small insertionsand deletions must be accurately identified. There must be internalcontrols against false-positive results and amenability to highthroughput automation. The LDR process of the present invention is ableto meet all of these objectives.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a flow diagram depicting a PCR/LDR process for detection ofcancer-associated mutations by electrophoresis or capture on anaddressable array where wild-type allele-specific oligonucleotide probesare excluded from the LDR phase to avoid overwhelming signal fromminority mutant target and no marker is added to the LDR phase.

FIG. 2 is a flow diagram depicting a PCR/LDR process for detection ofcancer-associated mutations by electrophoresis or capture on anaddressable array where wild-type allele-specific oligonucleotide probesare excluded from the LDR phase to avoid overwhelming signal fromminority mutant target and a marker is added to the LDR phase.

FIG. 3 is a flow diagram depicting a PCR/LDR process for detection ofcancer-associated mutations by electrophoresis or capture on anaddressable array where wild-type allele-specific oligonucleotide probesare utilized in the LDR phase at low levels and/or are modified to yieldless ligation product corresponding to the majority target. Thisprevents the signal from minority mutant target from being overwhelmed.

FIG. 4 is a schematic diagram depicting the PCR/LDR process of FIG. 1using electrophoresis to separate ligation products.

FIG. 5 is a schematic diagram depicting the PCR/LDR process of FIG. 2using electrophoresis to separate ligation products.

FIG. 6 is a schematic diagram depicting the PCR/LDR process of FIG. 3using electrophoresis to separate ligation products.

FIG. 7 is a schematic diagram depicting the PCR/LDR process of FIG. 1using an addressable array.

FIG. 8 is a schematic diagram depicting the PCR/LDR process of FIG. 2using an addressable array.

FIG. 9 is a schematic diagram depicting the PCR/LDR process of FIG. 3using an addressable array.

FIG. 10 relates to the construction of the Themus thermophilus DNAligase mutants at amino acid residue 294 using site-specificmutagenesis.

FIG. 11 shows the site-directed mutagenesis of possible active siteregions of Tth ligase. The horizontal bar represents the full-length TthDNA ligase protein with the arrow indicating the C-terminal end. Darkhatched bars represent regions with strong homology between Tth (i.e.Thermus thermophilus) DNA ligase and E. coil ligase, while the lighthatched bars indicate regions with less homology. Amino acidsubstitutions produced by site-directed mutagenesis at K118, D120, K294,R337, G339, C412, C415, C428, and C433 are indicated. Amino acidresidues which are identical among known NAD⁺-dependent ligases areunderlined.

FIGS. 12A-B show the primers for making the mutant ligases of FIG. 11.These primers bear the following sequence numbers: Primer a isidentified as SEQ. ID. No. 1(JL501) and SEQ. ID. No. 78 (JL505); Primerb is identified as SEQ. ID. No. 2 (JL503R), SEQ. ID. No. 79 (JL507R),SEQ. ID. No. 80 (JL537R), SEQ. ID. No. 81 (JL535R), SEQ. ID. No. 82(JL525R), SEQ. ID. No. 83 (JL527R), SEQ. ID. No. 84 (JL529R), SEQ. ID.No. 85 (JL531 R), and SEQ. ID. No. 86 (JL533R); Primer c is identifiedas SEQ. ID. No. 3 (JL502), SEQ. ID. No. 87 (JL509), SEQ. ID. No. 88(JL506), SEQ. ID. No. 89 (JL536), SEQ. ID. No. 90 (JL534), SEQ. ID. No.91 (JL524), SEQ. ID. No. 92 (JL526), SEQ. ID. No. 93 (JL528), SEQ. ID.No. 94 (JL530), and SEQ. ID. No. 95 (JL532); Primer d is identified asSEQ. ID. No. 4 (JL504R), SEQ. ID. No. 96 (JL508R), and SEQ. ID. No. 97(JL518R); Primer b1 is identified as SEQ. ID. No. 5; Primer c1 isidentified as SEQ. ID. No. 6; Primer b2 is identified as SEQ. ID. No. 8;and Primer c2 is identified as SEQ. ID. No. 7.

FIG. 13 shows a schematic representation of oligonucleotides used forligation assays. Probe sequences were derived from human eukaryoticprotein synthesis initiation factor eIF-4E (Rychlik, W., et al., Proc.Nati. Acad. Sci. USA, 84:945-49 (1987), which is hereby incorporated byreference). This random eukaryotic DNA sequence was chosen to avoid anyfalse signal arising from bacterial DNA contamination in partiallypurified mutant Tth DNA ligase preparations. The melting temperature ofprobes were predicted using the nearest neighbor thermodynamic method(Breslauer, K. J., et al., Proc. Natl. Acad. Sci. USA, 83:3746-3750(1986), which is hereby incorporated by reference) (OLIGO 4.0 program,National Biosciences Inc., Plymouth, Minn.). FIG. 13A and FIG. 13Brepresent the formation of nicked DNA duplex using one of the templatestrands, ALg, as an example. Shown in FIG. 13A, 4 different nicked DNAsubstrates are formed by annealing the common fluorescently labeledoligonucleotide, com5F, (SEQ. ID. No. 9) and one of the discriminatingoligos RP5′A (SEQ. ID. No. 10), RP5′C (SEQ. ID. No. 11), RP5′G (SEQ. ID.No. 12), RP5′T (SEQ. ID. No. 13) to the template strand, ALg (SEQ. ID.No. 14)). In the FIG. 13B, 4 different nicked DNA substrates are formedby annealing the fluorescently labeled oligonucleotide, com3F (SEQ. ID.No. 15), and one of the discriminating oligos (LP3′A (SEQ. ID. No. 16),LP3′C (SEQ, ID. No. 17), LP3′G (SEQ. ID. No. 18), LP3′T (SEQ. ID. No.19)) to the template strand, ALg (SEQ. ID. No. 14). The full set of all16 combinations of match and mismatch base pairing are thus formed byusing ALg (SEQ. ID. No. 14), GLg (SEQ. ID. No. 20), TLg (SEQ. ID. No.21), and CLg (SEQ. ID. No. 22) (shown in FIG. 13C) as the templatestrand, which vary at the underlined base. Products formed by ligationto the com-non fluorescently labeled probe can be discriminated by sizeon denaturing polyacrylamide gel due to the incorporation of differentlength of “A” tails.

FIG. 14 shows the sequences for the probes used to make theoligonucleotides of FIG. 13.

FIGS. 15A-E show the fidelity of nick closure by Tth DNA ligase at the3′-side of the nick. The ligase substrate (nicked DNA duplex), shown inFIG. 15A, is formed by annealing the discriminating oligonucleotideLP3′(A, C, G, or T) with a phosphorylated common oligonucleotide(3′-fluorescently labeled, com3F) on the template strand. Thediscriminating base “N” on the 3′-side of the discriminatingoligonucleotide, and the “n” in a template strand were varied to giveall 16 possible combinations of base-paring. “A_(m)” represents the “A”tail at the 5′-end of a discriminating oligonucleotide. Reactions werecarried out in 40 μl mixture containing 1 mM NAD⁺, 12.5 nM (500 fmoles)of nicked DNA duplex substrates and 0.125 nM (5 fmoles)Tth DNA ligase at65° C. Aliquots (5 μl) were removed at 0 hr, 2 hr, 4 hr, 6 hr, 8 hr, and23 hr and separated on denaturing polyacrylamide gels. Data was analyzedusing Genescan version 1.2 software. Results are plotted usingDeltagraph Pro3 Software. FIGS. 15B-E represent results obtained withthe same discriminating oligo, but with a different template strand. Inpanel 3′-A (FIG. 15B), the discriminating oligonucleotide was LP3′A. A-T(♦), A-C (Δ), A-A (∇), and A-G (∘) represent DNA substrates containingTLg, CLg, ALg, and GLg as the template strand, respectively. In panel3′-G (FIG. 15D), the discriminating oligonucleotide was LP3′G. G-C (♦),G-T (Δ), G-A (∇), and G-G (∘) represent DNA substrates with CLg, TLg,ALg, and GLg as the template strand, respectively. In panel 3′-C (FIG.15C), LP3′C was the discriminating oligonucleotide. C-G (♦), C-A (Δ),C-T (∇), and C-C (∘) indicate DNA substrates containing GLg, ALg, TLg,and CLg as the template strand, respectively. In panel 3′-T (FIG. 15E),LP3′T was the discriminating oligonucleotide. T-A (♦), T-G (Δ), T-T (∇),and T-C (∘) represent DNA substrates containing ALg, GLg, TLg, or CLg asthe template strand, respectively.

FIGS. 16 A-E show the fidelity of nick closure by a thermostable DNAligase at the 5′-side of the nick. Reaction conditions were the same asin FIG. 15 except that different discriminating and commonoligonucleotides were used. The discriminating base was on the 5′-sideof the nick (phosphorylated oligonucleotides RP5′A, C, G, or T), whilethe common oligonucleotide was on the 3′-side of the nick, and was5′-labeled with FAM (i.e. 6-carboxyfluorescein, fluorescent dye used insequencing and mutation detection). See FIG. 16A. In panel 5′-A (FIG.16B), the discriminating oligonucleotide was RP5′A. A-T (♦), A-C (Δ),A-A (∇), and A-G (∘) represent DNA substrates containing TLg, CLg, ALg,and GLg as the template strand, respectively. In panel 5′-G (FIG. 16D),the discriminating oligonucleotide was RP5′G. G-C (♦), G-T (Δ), G-A (∇),and G-G (∘) represent DNA substrate with CLg, TLg, ALg, and GLg as thetemplate strand, respectively. In panel 5′-C (FIG. 16C), RP5′C was thediscriminating oligonucleotide. C-G (♦), C-A (Δ), C-T (∇), and C-C (∘)indicate DNA substrates containing GLg, ALg, TLg, and CLg as thetemplate strand, respectively. In panel 5′-T (FIG. 16E), RP5′T was thediscriminating oligonucleotide. T-A (♦), T-G (Δ), T-T (∇), and T-C (∘)represent DNA substrates containing ALg, GLg, TLg, or CLg as thetemplate strand, respectively.

FIGS. 17A-C are diagrams of oligonucleotide probes containing a baseanalogue and mismatch in the third position on the 3′ side of the nick.

FIG. 18 shows a table for improving the fidelity of Thermus thermophilusligase. Sequences of probes LP3′C, LP3′T, and Corn 3F are shown in FIG.14. Sequences for other discriminating probes are as the follows:

SLP3′C: 5′ TACGTCTGCGGTGTTGCGTC 3′ (SEQ. ID. No. 23).

SLP3′T: 5′ CGTCTGCGGTGTTGCGTT 3′ (SEQ. ID. No. 24).

SLP3′ATC: 5′ ATGCGTCTGCGGTGTTGCATC 3′ (SEQ. ID. No. 25).

SLP3′ATT: 5′ GCGTCTGCGGTGTTGCATT 3′ (SEQ. ID. No. 26).

SLP3′QTC: 5′ AAATGCGTCTGCGGTGTTGCQTC 3′ (SEQ. ID. No. 27)

SLP3′QTT: 5′ ATGCGTCTGCGGTGTTGCQTT 3′ (SEQ. ID. No. 28)

Base “N” in the discriminating oligonucleotide represents either C or T.“Q” indicates the Q base analogue. The template strand for allsubstrates tested except those containing Q base analogues is GLg, andits sequence is shown in FIG. 14. The template strand in substratescontaining Q base analogues is GLg.m3A which differs from GLg at asingle site shown as bold. Initial rates of ligation (fmol/min) werecalculated as the slope of the linear graph with the X-axis as the timein min., and the y-axis as the amount of products in fmol. Ligationfidelity of Tth DNA ligase is defined as a ratio of the initial rate ofperfect match ligation over the initial rate of mismatch ligation. FIG.17 also shows the sequences for primers SLP3′TTC (SEQ. ID. No. 29),SLP3′TTT (SEQ. ID. No. 30), Corn 610-3′F (SEQ. ID. No. 31), GLg.m3A(SEQ. ID. No. 32), GLg.m3A.Rev (SEQ. ID. No. 33), ALg.m3A (SEQ. ID. No.34), ALg.m3A.Rev (SEQ. ID. No. 35), GLg. (SEQ. ID. No. 36), GLg.Rev(SEQ. ID. No. 37), SLP3′GTC (SEQ. ID. No. 38), SLP3′GTT (SEQ. ID. No.39), GLg.m3T (SEQ. ID. No. 40), and GLg.m3T.Rev (SEQ. ID. No. 41).

FIG. 19 shows the primers used for quantitative detection of single-basemutations in an excess of normal DNA by either wild-type or K294R TthDNA ligase. LDR reactions contained 12.5 nM (250 fmole) of themismatched template (GLg.m3A and GLg.m3A.rev=Normal template),containing from 0 to 2.5 nM (50 fmole) of perfect matched template(ALg.m3A and ALg.m3A.rev=Cancer template) in the presence of 25 fmol ofeither purified wild-type or mutant enzyme K294R. Each reaction wascarried out in a 20 μl mixture containing 20 mM Tris-HCl, pH 7.6; 10 mMMgCl₂; 100 mM KCl; 10 mM DTT; 1 mM NAD⁺; 25 nM (500 fmol) of the twoshort detecting primers and mixtures of templates. The reaction mixturewas heated in GeneAmp 9600 (Perkin Elmer) for 15 sec. at 94° C. beforeadding 25 fmol of the wild-type or mutant Tth DNA ligase. Afterincubation with the enzymes for another 30 sec at 94° C., LDR reactionswere run for 15 sec at 94° C., and 4 min. at 65° C. per cycle for 20cycles. Reactions were completely stopped by chilling the tubes in anethanol-dry ice bath, and adding 0.5 μl of 0.5 mM EDTA. Aliquots of 2.5μl of the reaction products were mixed with 2.5 μl of loading buffer(83% Formamide, 8.3 mM EDTA, and 0.17% Blue Dextran) and 0.5 μl GeneScanRox-1000 molecular weight marker, denatured at 94° C. for 2 min, chilledrapidly on ice prior to loading on an 8 M urea-10% polyacrylamide gel,and electrophoresed on an ABI 373 DNA sequencer at 1400 volts.Fluorescent ligation products were analyzed and quantified using the ABIGeneScan 672 software.

FIGS. 20A-B show the LDR oligonucleotide probes (FIG. 20A) and templatesequences (FIG. 20B) for a T:G mismatch in an excess of normal DNA byeither wild-type or mutant K294R Tth DNA ligase. GLg.m3A and GLg.m3A.revrepresent the mismatched template (Normal template), whereas ALg.m3A andALg.m3A.rev represent the perfect matched template (Cancer template);Primers SLP3′TTT represents the Normal primer for the perfect matched(Cancer) template; whereas SLP3′TTC represents the Normal primer for themismatched (Normal) template. Similarly, experiments with the Q-analoguefor a T:G mismatch use primers SLP3′Q₂TT (SEQ. ID. No. 42) andSLP3′Q₁₈TT (SEQ. ID. No. 43) as Normal primers for the matched (Cancer)template. The remaining sequences in FIG. 20 bear the SEQ. ID. Nos. setforth with respect to FIG. 17.

FIGS. 21A-B show the quantitative detection of single-base mutations (aT:G mismatch) in an excess of normal DNA by either wild-type or K294RTth DNA ligase. The amount of LDR product formed when 0, 0.025 nM (0.5fmol), 0.05 nM (1 fmol), 0.125 nM (2.5 fmol), 0.25 nM (5 fmol), 1.25 nM(25 fmol), and 2.5 nM (50 fmol) of “Cancer” template was used incombination with 12.5 nM (250 fmol) of the “Normal” template. Thereaction was carried out in the presence of 25 nM (500 fmol) of regularprimers (SLP3′TTT and Corn 610 3′F), and 1.25 nM (25 fmol) of thewild-type or K294R mutant enzymes. The oligonucleotide probes used inthis reaction create a T:G mismatch on the “Normal” template and a T:Amatch on the “Cancer” template at the ligation junction. LDR reactionswere run for 15 sec at 94° C., and 4 min. at 65° C. per cycle for 20cycles. Reactions were completely stopped by chilling the tubes in anethanol-dry ice bath and adding 0.5 μl of 0.5 mM EDTA. Aliquots of 2.5μl of the reaction products were mixed with 2.5 μl of loading buffer(83% Formamide, 8.3 mM EDTA, and 0.17% Blue Dextran) and 0.5 μl GeneScanRox-1000 molecular weight marker, denatured at 94° C. for 2 min, chilledrapidly on ice prior to loading on an 8 M urea-10% polyacrylamide gel,and electrophoresed on an ABI 373 DNA sequencer at 1400 volts.Fluorescent ligation products were analyzed and quantified using the ABIGeneScan 672 software. The table (FIG. 21B) describes the raw data forgraph (FIG. 21A). The data were analyzed and parameters of anexponential equation were fit to the data using the Deltagraph Pro 3.5.software. The X-axis indicated the different amounts of Cancer Templatein 12.5 nM (250 fmol) of the Normal Template, while the Y-axis indicatedthe amount of LDR product generated. (▪) represents 1.25 nM (25 fmol) ofthe wild-type enzyme whereas () represents 1.25 nM (25 fmol) of mutantK294 R enzyme.

FIGS. 22A-B show the signal-to-noise ratio of the LDR product withdifferent concentrations of mutant template in normal DNA. Thesingle-base mutation template (“Cancer”) was diluted into 12.5 nM (250fmol) of the “Normal” template, and assayed using 0.125 nM (25 fmol) ofeither wild-type or mutant K294R Tth DNA ligase. The oligonucleotideprobes used in this reaction create a T:G mismatch on the “Normal”template and a T:A match on the “Cancer” template at the ligationjunction. The signal-to-noise ratio is defined as the ratio of theamount of product formed with “Cancer” Template in the presence of“Normal” template (12.5 nM=250 fmol template) to the amount of productformed by the same amount of Normal template alone. The table (FIG. 22B)describes the raw data for the graph (FIG. 22A). The data were analyzedand parameters of an exponential equation fit to the data using theDeltagraph Pro 3.5. software. The X-axis displayed different amounts of“Cancer” template in 12.5 nM (250 fmol) of the “Normal” template, whilethe Y-axis indicated the amount of LDR product generated. (▪) represents1.25 nM (25 fmol) of the wild-type enzyme whereas () represents 1.25 nM(25 fmol) of mutant K294 R enzyme.

FIGS. 23A-B show the amount of LDR product formed when 0, 0.005 nM (0.1fmol), 0.0125 nM (0.25 fmol), 0.025 nM (0.5 fmol), 0.05 nM (1.0 fmol),0.125 nM (2.5 fmol), 0.25 nM (5 fmol), and 0.5 nM (10 fmol) of Normaltemplate (Glg.m3A and GLg.m3.Arev), are mixed with 25 nM (500 fmol) ofregular primers (SLP3′TTC and Com 610 3′F), and 1.25 nM (25 fmol) of thewild-type or mutant enzymes. The primers used in this reaction create aC:G match on the “Normal” template at the ligation junction. LDRreactions were run for 15 sec at 94° C., and 4 min. at 65° C. per cyclefor 20 cycles. Reactions were completely stopped by chilling the tubesin an ethanol-dry ice bath, and adding 0.5 μl of 0.5 mM EDTA. Aliquotsof 2.5 μl of the reaction products were mixed with 2.5 μl of loadingbuffer (83% Formamide, 8.3 mM EDTA, and 0.17% Blue Dextran) and 0.5 μlGeneScan Rox-1000 molecular weight marker, denatured at 94° C. for 2min, chilled rapidly on ice prior to loading on an 8 M urea-10%polyacrylamide gel, and electrophoresed on an ABI 373 DNA sequencer at1400 volts. Fluorescent ligation products were analyzed and quantifiedusing the ABI GeneScan 672 software. The table (FIG. 23B) describes theraw data for the graph (FIG. 23A). The data were analyzed and parametersof an exponential equation fit to the data using the Deltagraph Pro 3.5.software. The X-axis indicated the different amounts of Cancer Templatein 12.5 nM (250 fmol) of the Normal Template, while the Y-axis indicatedthe amount of LDR product generated. (▪) represents 1.25 nM (25 fmol) ofthe wild-type enzyme whereas () represents 1.25 nM (25 fmol) of mutantK294 R enzyme.

FIG. 24 shows nucleotide analogue containing primers used for assayingligase fidelity. Four different conditions were used to assess thefidelity of the wild-type and mutant Tth DNA ligase in a typical LDRassay. Each reaction was carried out in a 20 μl mixture containing 20 mMTris-HCl, pH 7.6; 10 mM MgCl₂; 100 mM KCl; 10 mM DTT; 1 mM NAD⁺; 25 nM(500 fmol) of the two short detecting primers and 12.5 nM (250 fmol) ofthe normal template when used alone, or 125 nM (2.5 fmol), and 0.5 nM(10 fmol), of the cancer template when used together with the normaltemplate in a ratio of 1:100 and 1:25, respectively. The oligonucleotideprobes used in this reaction create a T:G mismatch on the “Normal”template and an T:A match on the “Cancer” template at the ligationjunction. In addition, oligonucleotide probes SLP3′QTT create a Q₂:A orQ₁₈:T pairing at the 3rd position from the 3′ end. The reaction mixturewas heated in a GeneAmp 9600 thermocycler (Perkin Elmer) for 1.5 sec. at94° C. before adding 25 fmol of the wild-type and mutant Tth DNA ligase.After incubation with the enzymes for another 30 sec, LDR reactions wererun for 15 sec at 94° C., and 4 min. at 65° C. per cycle for 20 cycles.Reactions were completely stopped by chilling the tubes in anethanol-dry ice bath, and adding 0.5 μl of 0.5 mM EDTA. 2.5 μl ofreaction product was mixed with 2.5 μl of loading buffer (83% Formamide,8.3 mM EDTA, and 0.17% Blue Dextran) and 0.5 μl Gene Scan Rox-1000molecular weight marker, denatured at 94° C. for 2 min, chilled rapidlyon ice prior to loading on an 8 M urea-10% polyacrylamide gel, andelectrophoresed on an ABI 373 DNA sequencer. Fluorescent labeledligation products were analyzed and quantified using the ABI Gene Scan672 software.

FIGS. 25A-D show different forms of oligonucleotide probes withnucleotide analogues for the LDR phase of the PCR/LDR process of thepresent invention. The probes in FIG. 25A, B, C, and D with thenucleotide analog Q are designated SEQ. ID. Nos. 44, 45, 46, and 47,respectively. In each of these figures, the target DNA sequence is thesame and is designated SEQ. ID. No. 48.

FIG. 26 shows primers used for quantitative detection of single-basemutations in an excess of normal DNA by either wild-type or K294R TthDNA ligase (C:A mismatch). LDR reactions contained 12.5 nM (250 fmole)of the mismatched template (ALg.m3A and ALg.m3A.rev=Normal template)containing from 0 to 2.5 nM (50 fmole) of perfect matched template(GLg.m3A and GLg.m3A.rev=Cancer template), in the presence of 25 fmol ofeither purified wild-type or mutant enzyme K294R. Each reaction wascarried out in a 20 μl mixture containing 20 mM Tris-HCl, pH 7.6; 10 mMMgCl₂; 100 mM KCl; 10 mM DTT; 1 mM NAD⁺; 25 nM (500 fmol) of the twoshort detecting oligonucleotides probes and mixtures of templates. Theprobes used in this reaction create a C:A mismatch on the “Normal”template and an C:G match on the “Cancer” template at the ligationjunction. The reaction mixture was heated in GeneAmp 9600 (Perkin Elmer)for 1.5 sec. at 94° C. before adding 25 fmol of the wild-type or mutantTth DNA ligase. After incubation with the enzymes for another 30 sec,LDR reactions were run for 15 sec at 94° C., and 4 min. at 65° C. percycle for 20 cycles. Reactions were completely stopped by chilling thetubes in an ethanol-dry ice bath, and adding 0.5 μl of 0.5 mM EDTA.Aliquots of 2.5 μl of the reaction products were mixed with 2.5 μl ofloading buffer (83% Formamide, 8.3 mM EDTA, and 0.17% Blue Dextran) and0.5 μl GeneScan Rox-1000 molecular weight markers denatured at 94° C.for 2 min, chilled rapidly on ice prior to loading on an 8 M urea-10%polyacrylamide gel, and electrophoresed on an ABI 373 DNA sequencer at1400 volts. Fluorescent ligation products were analyzed and quantifiedusing the ABI GeneScan 672 software.

FIGS. 27A-B show the quantitative detection of single-base mutations (aC:A mismatch) in an excess of normal DNA by either wild-type or K294RTth DNA ligase. The amount of LDR product formed when 0, 0.025 nM (0.5fmol), 0.05 nM (1 fmol), 0.125 nM (2.5 fmol), 0.25 nM (5 fmol), 1.25 nM(25 fmol), and 2.5 nM (50 fmol) of “Cancer” (i.e. Glg.m3A/Glg.m3A.rev)template was used in combination with 12.5 nM (250 fmol) of the “Normal”template (i.e. ALg.m3A/ALg.m3A.rev). The reaction was carried out in thepresence of 25 nM (500 fmol) of regular oligonucleotide probes (SLP3′TTCand Com 610 3′F), and 1.25 nM (25 fmol) of the wild-type or mutantenzymes. The oligonucleotide probes used in this reaction create a C:Amismatch on the “Normal” template and a C:G match on the “Cancer”template at the ligation junction. LDR reactions were run for 15 sec at94° C. and 4 min. at 65° C. per cycle for 20 cycles. Reactions werecompletely stopped by chilling the tubes in an ethanol-dry ice bath, andadding 0.5 μl of 0.5 mM EDTA. Aliquots of 2.5 μl of the reactionproducts were mixed with 2.5 μl of loading buffer (83% Formamide, 8.3 mMEDTA, and 0.17% Blue Dextran) and 0.5 μl GeneScan Rox-1000 molecularweight marker, denatured at 94° C. for 2 min, chilled rapidly on iceprior to loading on an 8 M urea-10% polyacrylamide gel, andelectrophoresed on an ABI 373 DNA sequencer. Fluorescent ligationproducts were analyzed and quantified using the ABI GeneScan 672software. The table (FIG. 26B) describes the raw data for the graph(FIG. 26A). The data were analyzed and parameters of an exponentialequation fit to the data using the Deltagraph Pro 3.5. software. TheX-axis indicated the different amounts of Cancer Template in 12.5 nM(250 fmol) of the Normal Template, while the Y-axis indicated the amountof LDR product generated. (▪) represents 1.25 riM (25 fmol) of thewild-type enzyme whereas () represents 1.25 nM (25 fmol) of mutant K294R enzyme.

FIGS. 28A-B show the signal-to-noise ratio of the LDR product withdifferent concentrations of the single-base mutation template (“Cancer”)in combination with 12.5 nM (250 fmol) of the “Normal” template with0.125 nM (25 fmol) by either wild-type or mutant K294R Tth DNA ligase.The signal-to-noise ratio is described as the ratio of the amount ofproduct formed with varying concentrations of Cancer Template in thepresence of 12.5 nM (250 fmol) of Normal template to the amount ofproduct formed by 12.5 nM (250 fmol) of Normal template alone. Theoligonucleotide probes (SLp3′ TTC and Com 6103′ F) used in this reactioncreate a C:A mismatch on the “Normal” template (ALg.m3A/ALg.m3A.rev) andan C:G match on the “Cancer” template (GLg.m3A/GLg.m3A.rev) at theligation junction The table (FIG. 28B) describes the raw data for thegraph (FIG. 28A). The data were analyzed and parameters of anexponential equation fit to the data using the Deltagraph Pro 3.5.software. The X-axis displayed different amounts of “Cancer” template in12.5 nM (250 fmol) of the “Normal” template, while the Y-axis indicatedthe amount of LDR product generated. (▪) represents 1.25 riM (25 fmol)of the wild-type enzyme whereas () represents 1.25 nM (25 fmol) ofmutant K294 R enzyme.

FIGS. 29A-B show the amount of LDR product formed when 0, 0.005 nM (0.1fmol), 0.0125 nM (0.25 fmol), 0.025 nM (0.5 fmol), 0.05 nM (1.0 fmol),0.125 nM (2.5 fmol), 0.25 nM (5 fmol), and 0.5 nM (10 fmol) of Normaltemplate (i.e. ALg.m3A/ALg.m3A.rev) alone, are reacted with 25 nM (500fmol) of regular primers (SLP3′TTT and Corn 610 3′F), and 1.25 nM (25fmol) of the wild-type or mutant enzymes. LDR reactions were run for 15sec at 94° C. and 4 min. at 65° C. per cycle for 20 cycles. Theoligonucleotide probes used in this reaction create a T:A match on the“Normal” template at the ligation junction. Reactions were completelystopped by chilling the tubes in an ethanol-dry ice bath, and adding 0.5μl of 0.5 mM EDTA. Aliquots of 2.5 μl of the reaction products weremixed with 2.5 μl of loading buffer (83% Formamide, 8.3 mM EDTA, and0.17% Blue Dextran) and 0.5 μl GeneScan Rox-1000 molecular weightmarker, denatured at 94° C. for 2 min, chilled rapidly on ice prior toloading on an 8 M urea-10% polyacrylamide gel, and electrophoresed on anABI 373 DNA sequencer at 1400 volts. Fluorescent ligation products wereanalyzed and quantified using the ABI GeneScan 672 software. The table(FIG. 29B) describes the raw data for the graph (FIG. 29A). The datawere analyzed and parameters of an exponential equation fit to the datausing the Deltagraph Pro 3.5. software. The X-axis indicated the amountsof the Normal Template used, while the Y-axis indicated the amount ofLDR product generated. (▪) represents 1.25 nM (25 fmol) of the wild-typeenzyme whereas () represents 1.25 nM (25 fmol) of mutant K294 R enzyme.

FIGS. 30A-C show a scheme for PCR/LDR detection of mutations in codons12, 13, and 61 of K-ras. At the top of the drawing (FIG. 30A) is aschematic representation of the chromosomal DNA containing the K-rasgene. Exons are shaded and the position of codons 12, 13, and 61 shown.Exon-specific primers are used to amplify selectively K-ras DNA flankingthese three codons. The middle (FIG. 30B) and bottom (FIG. 30C) of thediagram gives a schematic representation of primer design for LDRdetection of all possible amino acid changes in codons 12, 13, and 61.For example, codon 12 (GGT) may mutate to GAT, GCT, or GTT.Allele-specific LDR oligonucleotide probes contain the discriminatingbase on the 3′ end and a fluorescent label on the 5′ end. Commonoligonucleotides are phosphorylated on the 5′ end and contain a poly-Atail and blocking group on the 3′ end. Different mutations aredistinguished by separating the products on a polyacrylamide gel. Notethat LDR oligonucleotide probes used for detecting mutations at codon 12may interfere with hybridization of oligonucleotide probes used todetect mutations at codon 13. It will be necessary to determineexperimentally if these probes can correctly identify mutant signal inthe presence of the other LDR probes.

FIGS. 31A-B provide the name and sequence of K-ras LDR oligonucleotideprobes used to detect different mutations at codons 12, 13, and 61 in atypical LDR reaction in an excess of normal DNA by either wild-type ormutant K294R 7th DNA ligase. FIGS. 31A-B show the sequences for thefollowing primers: Fam-K-ras c12.2D (SEQ. ID. No. 49), Tet-K-ras c12.2A(SEQ. ID. No. 50), Fam-K-ras c12.2V (SEQ. ID. No. 51), K-ras c12 Com-2(SEQ. ID. No. 52), Tct-K-ras c12.1S (SEQ. ID. No. 53), Fam-K-ras c12.1R(SEQ. ID. No. 54), Tet-K-ras c12.1C (SEQ. ID. No. 55), K-ras c12 Com-1(SEQ. ID. No. 56), Fam-K-ras c13.4D (SEQ. ID. No. 57), Tet-K-ras c13.4A(SEQ. ID. No. 58), Fam-K-ras c13.4V (SEQ. ID. No. 59), K-ras c13 Com-4(SEQ. ID. No. 60), Tet-K-ras c13.3S (SEQ. ID. No. 61), Fam-K-ras c13.3R(SEQ. ID. No. 62), Tet-K-ras c13.3C (SEQ. ID. No. 63), K-ras c13 Com-3(SEQ. ID. No. 64), Tet-K-ras c61.7HT (SEQ. ID. No. 65), Fam-K-rasc61.7HC (SEQ. ID. No. 66), K-ras Com-7 (SEQ. ID. No. 67), Tet-K-rasc61.6R (SEQ. ID. No. 68), Fam-K-ras c61.6P (SEQ. ID. No. 69), Tet-K-rasc61.6P (SEQ. ID. No. 70), K-ras Com-6 (SEQ. ID. No. 71), Fam-K-rasc61.5K (SEQ. ID. No. 72), Tet-K-ras c61.5E (SEQ. ID. No. 73), and K-rasCom-5 (SEQ. ID. No. 74).

FIGS. 32 and 33A-B show the quantitative detection of a Gly→Asp mutation(C:A mismatch) in Codon 12 of the K-ras gene (G12D) in an excess ofnormal K-ras sequence by either wild-type or K294R Tth DNA ligase. Theeleven lanes on the left of gel # mk960423 (#1-11) of FIG. 32 representdata obtained when using wild-type Tth DNA ligase, while the elevenlanes on the right (#13-23) of FIG. 32 represent data obtained whenusing the mutant Tth DNA ligase, K294R. The first three lanes in eachcase are negative controls without any added mutant K-ras sequence. Thenext eight lanes depict the amount of LDR product formed when 5 nM (100fmol), 2 nM (40 fmol), 0.8 nM (20 fmol), 0.4 nM (8 fmol), 0.2 nM (4fmol), 0.1 nM (2.0 fmol), 0.05 nM (1 fmol), and 0.025 nM (0.5 fmol) ofmutant K-ras template, respectively, was used in combination with 100 nM(2000 fmol) of the wild-type K-ras DNA. The reaction was carried out inthe presence of 25 nM (500 fmol) of one discriminating oligonucleotideprobes (Fam-K-ras c12.2D, and a common primer K-ras c12 Com-2) and 5 nM(100 fmol) of the wild-type or K294R mutant enzymes. The LDR probe fordetecting the Gly→Asp mutation used in this reaction creates a C:Amismatch on the wild-type template and a T:A match on the Gly→Asp mutantK-ras template at the ligation junction. PCR reactions were run for 15sec at 94° C., 1 min at 55° C., and 1 min. (+3 sec/cycle) at 72° C. percycle for 30 cycles. LDR reactions were run for 15 sec at 94° C., and 4min. at 65° C. per cycle for 20 cycles. Reactions were completelystopped by chilling the tubes in an ethanol-dry ice bath, and adding 0.5μl of 0.5 mM EDTA. Aliquots of 2.5 μl of the reaction products weremixed with 2.5 μl of loading buffer (83% Formamide, 8.3 mM EDTA, and0.17% Blue Dextran) and 0.5 μl GeneScan TAMRA 350 molecular weightmarker, denatured at 94° C. for 2 min, chilled rapidly on ice prior toloading on an 8 M urea-10% polyacrylamide gel, and electrophoresed on anABI 373 DNA sequencer at 1400 volts. Fluorescent ligation products werequantified using the ABI GeneScan 672 software. The table (FIG. 33B)describes the raw data for the graph (FIG. 33A). The data were analyzedand parameters of an exponential equation fit to the data using theDeltagraph Pro 3.5. software. The X-axis indicated the different amountsof G1 2D template in 100 nM (2000 fmol) of the wild-type template, whilethe Y-axis indicated the amount of LDR product generated. (▪) represents5 nM (100 fmol) of the wild-type enzyme whereas () represents 5 nM (100fmol) of mutant K294 R enzyme.

FIGS. 34A-B show the signal-to-noise ratio of the LDR product withdifferent concentrations of the K-ras gene (from 0.025 nM [0.5 fmol] to5 nM [100 fmol]) containing a single-base mutation (G12D) in combinationwith 100 nM (2000 fmol) of the wild-type K-ras template using 5 nM (100fmol) of either wild-type or mutant K294R Tth DNA ligase. The probesused in this reaction create a C:A mismatch on the wild-type templateand a T:A match on the G12D template at the ligation junction. Thesignal-to-noise ratio is defined as the ratio of the amount of productformed with G12D template in the presence of wild-type template (100 nM2000 fmol template) to the amount of product formed by the same amountof wild-type template alone. The table (FIG. 34B) describes the raw datafor the graph (FIG. 34A). The data were analyzed and parameters of anexponential equation fit to the data using the Deltagraph Pro 3.5.software. The X-axis displayed different amounts of G12D template in 100nM (2000 fmol) of wild-type template, while the Y-axis indicated theamount of LDR product generated. (▪) represents 5 nM (100 fmol) of thewild-type enzyme whereas () represents 5 nM (100 fmol) of mutant K294 Renzyme.

FIGS. 35 and 36A-B show the quantitative detection of a Gly→Val mutation(a C:T mismatch) in Codon 12 of the K-ras gene (G12V) in an excess ofnormal K-ras sequence by either wild-type or K294R Tth DNA ligase. Theeleven lanes on the left of gel # mk960405 (#1-11) (FIG. 35) representdata obtained when using wild-type Tth DNA ligase, while the elevenlanes on the right (#13-23) (FIG. 35) represent data obtained when usingthe mutant Tth DNA ligase, K294R. The first three lanes (FIG. 35) ineach case are negative controls without any added mutant K-ras sequence.The next eight lanes (FIG. 35) depict the amount of LDR product formedwhen 0.1 nM (2.0 fmol), 0.2 nM (4 fmol), 0.4 nM (8 fmol), 0.8 nM (20fmol), 2 nM (40 fmol), 4 nM (80 fmol), 5 nM (100 fmol), and 20 nM (200fmol) of mutant K-ras template was used in combination with 100 nM (2000fmol) of the wild-type K-ras template. The reaction was carried out inthe presence of 25 nM (500 fmol) of six discriminating probes (Tet-K-rasc12.2A, Tet-K-ras c12.1S, Tet-K-ras c12.1C, Fam-K-ras c12. IR, Fam-K-rasc12.2D, Fam-K-ras c12.2V); 75 nM (1500 fmol) of two common probes (K-rasc12 Com-2 and K-ras c12 Com-1) and 5 nM (100 fmol) of the wild-type orK294R mutant enzymes. Tet is tetrachlorinated-6-carboxyfluorescein;fluorescent dye used in sequencing/mutation detection, while Corn is anoligonucleotide probe using a 3′ amino modified C3CPG column. Thiscolumn is designed to produce a primary amine to the 3′ terminus of atarget oligonucleotide. The primary use of these 3′ amino modifiers arefor subsequent labelling as diagnostic probes and to generate anoligonucleotide resistant to the 3′ exonuclease activity. This set ofprobes is capable of detecting the presence of all six single-basemutations in Codon 12 of the K-ras gene in a multiplex reaction. The LDRprobe for detecting the Gly→Val mutation used in this reaction creates aC:T mismatch on the wild-type template and an A:T match on the Gly→Valmutant K-ras template at the ligation junction. PCR reactions were runfor 15 sec at 94° C., 1 min at 55° C., and 1 min. (+3 sec/cycle) at 72°C. per cycle for 30 cycles. LDR reactions were run for 15 sec at 94° C.,and 4 min. at 65° C. per cycle for 20 cycles. Reactions were completelystopped by chilling the tubes in an ethanol-dry ice bath, and adding 0.5μl of 0.5 mM EDTA. Aliquots of 2.5 μl of the reaction products weremixed with 2.5 μl of loading buffer (83% Formamide, 8.3 mM EDTA, and0.17% Blue Dextran) and 0.5 μl GeneScan TAMRA 350 molecular weightmarker, denatured at 94° C. for 2 min, chilled rapidly on ice prior toloading on an 8 M urea-10% polyacrylamide gel, and electrophoresed on anABI 373 DNA sequencer at 1400 volts. Fluorescent ligation products werequantified using the ABI GeneScan 672 software. The table (FIG. 36B)describes the raw data for the graph (FIG. 36A). The data were analyzedand parameters of an exponential equation fit to the data using theDeltagraph Pro 3.5. software. The X-axis indicated the different amountsof G12V template in 100 nM (2000 fmol) of the wild-type template, whilethe Y-axis indicated the amount of LDR product generated. (▪) represents5 nM (100 fmol) of the wild-type enzyme whereas () represents 5 nM (100fmol) of mutant K294 R enzyme.

FIGS. 37A-B show the signal-to-noise ratio of the LDR product in 26primer multiplex reaction with different concentrations of the K-rasgene (from 0.1 nM [2 fmol] to 10 nM [200 fmol]) containing a single-basemutation (G12V) in combination with 100 nM (2000 fmol) of the wild-typeK-ras template using 5 nM (100 fmol) of either wild-type or mutant K294RTth DNA ligase. The G12V specific probes used in this reaction create aC:T mismatch on the wild-type template and an A:T match on the G12Vtemplate at the ligation junction. The greatest background noise in thismultiplexed reaction was from probes designed to detect Q61R,representing a G:T mismatch, which was about 10-fold higher than fromprobes designed to detect G12D, i.e. representing a C:A mismatch. Forconsistency with other assays, the signal-to-noise ratio in thismultiplexed assay is defined as the ratio of the amount of productformed with G12V templates in the presence of wild-type template (100nM=2000 fmol template) to the amount of G12D LDR product formed by thesame amount of wild-type template alone (representing a C:A mismatch).The table (FIG. 37B) describes the raw data for the graph (FIG. 37A).The data were analyzed and parameters of an exponential equation fit tothe data using the Deltagraph Pro 3.5. software. The X-axis displayeddifferent amounts of “Cancer” template in 100 nM (2000 fmol) of the“Normal” template, while the Y-axis indicated the amount of LDR productgenerated. () represents 5 nM (100 fmol) of the wild-type enzymewhereas () represents 5 nM (100 fmol) of mutant K294 R enzyme.

FIGS. 38 and 39A-B show the quantitative detection of a Gly→Val mutation(a C:T mismatch) in Codon 12 of the K-ras gene in an excess of normalK-ras sequence by either wild-type or K294R Tth DNA ligase. The elevenlanes on the left of gel # mk960429 (#1-11) (FIG. 38) represent dataobtained when using wild-type Tth DNA ligase, while the eleven lanes onthe right (#13-23) (FIG. 38) represent data obtained when using themutant Tth DNA ligase, K294R. The first three lanes in each case arenegative controls without any added mutant K-ras sequence. The nexteight lanes depict the amount of LDR product formed when 0.1 nM (2.0fmol), 0.2 nM (4 fmol), 0.4 nM (8 fmol), 0.8 nM (10 fmol), 2 nM (20fmol), 4 nM (40 fmol), 5 nM (100 fmol), and 20 nM (200 fmol) of mutantK-ras template was used in combination with 100 nM (2000 fmol) of thewild-type K-ras template. The reaction was carried out in the presenceof 25 nM (500 fmol) of nineteen discriminating primers (Tet-K-rasc12.2A, Tet-K-ras c12.1S, Tet-K-ras c12.1C, Tet-K-ras c13.4A, Tet-K-rasc13.3S, Tet-K-ras c13.3C, Tet-K-ras c61.7HT, Tet-K-ras c61.6R, Tet-K-rasc61.5K, Tet-K-ras c61.6P, Fam-K-ras c12.1R, Fam-K-ras c12.2D, Fam-K-rasc12.2V, Fam-K-ras c13.4D, Fam-K-ras c13.4V, Fam-K-ras c13.3R, Fam-K-rasc61.7HC, Fam-K-ras c61.6L, Fam-K-ras c61.5K); 50 nM (1000 fmol) of twocommon probes (K-ras c61 Com-7 and K-ras c12 Com-5); and 75 nM (1500fmol) of five common primers (K-ras c12 Com-2, K-ras c12 Com-1, K-rasc13 Com-4, K-ras c13 Com-3, and K-ras c61 Com-6) and 5 nM (100 fmol) ofthe wild-type or K294R mutant enzymes. This set of probes is capable ofdetecting the presence of all nineteen mutations in Codons 12, 13, and61, of the K-ras gene in a multiplex reaction. The LDR probe fordetecting the Gly→Val mutation used in this reaction creates a C:Tmismatch on the wild-type K-ras template and an A:T match on the Gly→Valmutant K-ras template at the ligation junction. PCR reactions were runfor 15 sec at 94° C., 1 min at 55° C., and 1 min. (+3 sec/cycle) at 72°C. per cycle for 30 cycles. LDR reactions were run for 15 sec at 94° C.,and 4 min. at 65° C. per cycle for 20 cycles. Reactions were completelystopped by chilling the tubes in an ethanol-dry ice bath, and adding 0.5μl of 0.5 mM EDTA. Aliquots of 2.5 μl of the reaction products weremixed with 2.5 μl of loading buffer (83% Formamide, 8.3 mM EDTA, and0.17% Blue Dextran) and 0.5 μl GeneScan TAMRA 350 molecular weightmarker, denatured at 94° C. for 2 min, chilled rapidly on ice prior toloading on an 8 M urea-10% polyacrylamide gel, and electrophoresed on anABI 373 DNA sequencer at 1400 volts. Fluorescent ligation products werequantified using the ABI GeneScan 672 software. The table (FIG. 39B)describes the raw data for the graph (FIG. 39A). The data were analyzedand parameters of an exponential equation fit to the data using theDeltagraph Pro 3.5. software. The X-axis indicated the different amountsof G12V template in 100 nM (2000 fmol) of the wild-type template, whilethe Y-axis indicated the amount of LDR product generated. (▪) represents5 nM (100 fmol) of the wild-type enzyme whereas () represents 5 nM (100fmol) of mutant K294 R enzyme.

FIGS. 40A-B show the signal-to-noise ratio of the LDR product in a 26primer multiplex reaction with different concentrations of the K-rasgene (from 0.1 nM [2 fmol] to 10 nM [200 fmol]) containing a single-basemutation (G12V) in combination with 100 nM (2000 fmol) of the wild-typeK-ras template using 5 nM (100 fmol) of either wild-type or mutant K294RTth DNA ligase. The G12V specific probes used in this reaction create aC:T mismatch on the wild-type template and an A:T match on the G12Vtemplate at the ligation junction. The greatest background noise in thismultiplexed reaction was from probes designed to detect Q61R,representing a G:T mismatch, which was about 10-fold higher than fromprobes designed to detect G12D, i.e. representing a C:A mismatch. Forconsistency with our other assays, the signal-to-noise ratio in thismultiplexed assay is defined as the ratio of the amount of productformed with G12V templates in the presence of wild-type template (100nM=2000 fmol template) to the amount of G12D LDR product formed by thesame amount of wild-type template alone (representing a C:A mismatch).The table (FIG. 40B) describes the raw data for the graph (FIG. 40A).The data were analyzed and parameters of an exponential equation fit tothe data using the Deltagraph Pro 3.5. software. The X-axis displayeddifferent amounts of “Cancer” template in 100 nM (2000 fmol) of the“Normal” template, while the Y-axis indicated the amount of LDR productgenerated. (▪) represents 5 nM (100 fmol) of the wild-type enzymewhereas () represents 5 nM (100 fmol) of mutant K294 R enzyme.

FIGS. 41-42 show the quantitative detection of mutations in the K-rasgene by K294 mutant Tth DNA ligase. The first lane (FIG. 41) in gel #mk950513 is a negative control using wild-type K-ras DNA. The secondlane (FIG. 41) is a positive control which contains mutant Gly→Val K-rasDNA. The next twenty lanes (FIG. 41) represent a blind test of LDRreactions on twenty samples containing different mutant K-ras DNA. Thereactions were carried out in the presence of 25 nM (500 fmol) ofnineteen discriminating primers (Tet-K-ras c12.2A, Tet-K-ras c12.1S,Tet-K-ras c12.1C, Tet-K-ras c13.4A, Tet-K-ras c13.3S, Tet-K-ras c13.3C,Tet-K-ras c61.7HT, Tet-K-ras c61.6R, Tet-K-ras c61.5K, Tet-K-ras c61.6P,Fam-K-ras c12.1R, Fam-K-ras c12.2D, Fam-K-ras c12.2V, Fam-K-ras c13.4D,Fam-K-ras c13.4V, Fam-K-ras c13.3R, Fam-K-ras c61.7HC, Fam-K-ras c61.6L,Fam-K-ras c61.5K); 50 nM (1000 fmol) of two common probes (K-ras c61Com-7 and K-ras c12 Com-5); and 75 nM (1500 fmol) of five common probes(K-ras c12 Com-2, K-ras c12 Com-1, K-ras c13 Com-4, K-ras c13 Com-3, andK-ras c61 Corn-6) and 5 nM (100 fmol) of the wild-type or K294R mutantenzymes. This set of probes is capable of detecting the presence of allnineteen mutations in Codons 12, 13, and 61, of the K-ras gene in amultiplex reaction. Microdissected tissue was transferred to a PCR tube,exposed to xylene for 10 min, washed 3× in 95% ethanol, and desiccated.PCR reactions were run for 30 sec at 94° C., 1.5 min at 54° C., and 1min. at 72° C. per cycle for 35 cycles. LDR reactions were run for 15sec at 94° C., and 4 min. at 65° C. per cycle for 20 cycles. Reactionswere completely stopped by chilling the tubes in an ethanol-dry icebath, and adding 0.5 μl of 0.5 mM EDTA. Aliquots of 2.5 μl of thereaction products were mixed with 2.5 μl of loading buffer (83%Formamide, 8.3 mM EDTA, and 0.17% Blue Dextran) and 0.5 μl GeneScanTAMRA 350 molecular weight marker, denatured at 94° C. for 2 min,chilled rapidly on ice prior to loading on an 8 M urea-10%polyacrylamide gel, and electrophoresed on an ABI 373 DNA sequencer at1400 volts. Fluorescent ligation products were quantified using the ABIGeneScan 672 software. The data were analyzed and the results arepresented in FIG. 42 (called mutation) where they are compared with theresults determined by dideoxy-sequencing (expected mutation).

FIG. 43 is a table comparing 10 discordant samples from the PCR/LDRprocess described above with reference to FIGS. 41-42.

FIGS. 44 and 45A-B show the quantitative detection of different amountsof K-ras Normal template when varying amounts of wild-type probes wereused by either wild-type or K294R Tth DNA ligase. Amount of LDR productformed when 25 nM (500 fmol), 50 nM (1000 fmol), and 100 nM (2000 fmol)of the “Normal” template was reacted with 0.5 nM (10 fmol), 2.5 nM (50fmol), and 5 nM (100 fmol) of the wild type discriminating probe(Tet-K-ras c12.2 WtG) and common probe (K-ras c12 Com-2) in the presenceof 25 nM (500 fmol) of nineteen discriminating probes (Tet-K-ras c12.2A,Tet-K-ras c12.1S, Tet-K-ras c12.1C, Tet-K-ras c13.4A, Tet-K-ras c13.3S,Tet-K-ras c13.3C, Tet-K-ras c61.7HT, Tet-K-ras c61.6R, Tet-K-ras c61.5K,Tet-K-ras c61.6P, Fam-K-ras c12.1R, Fam-K-ras c12.2D, Fam-K-ras c 12.2V,Fam-K-ras c13.4D, Fam-K-ras c13.4V, Fam-K-ras c13.3R, Fam-K-ras c61.7HC,Fam-K-ras c61.6L, Fam-K-ras c61.5K); 50 nm (1000 fmol) of two commonprobes (K-ras c61 Com-7 and K-ras c12 Com-5); and 75 nm (1500 fmol) offive common probes (K-ras c12 Com-2, K-ras c12 Corn-1, K-ras c13 Com-4,K-ras c13 Com-3, and K-ras c61 Com-6) and 5 nm (100 fmol) of the wildtype or K294R mutant enzymes. LDR reactions were run for 15 sec at 94°C. and 4 min. at 65° C. per cycle for 20 cycles. The reactions werecompletely stopped by chilling the tubes in an ethanol-dry ice bath, andadding 0.5 μl of 0.5 mM EDTA. Aliquots of 2.5 μl of the reactionproducts were mixed with 2.5 μl of loading buffer (83% Formamide, 8.3 mMEDTA, and 0.17% Blue Dextran) and 0.5 μl GeneScan TAMRA 350 molecularweight marker, denatured at 94° C. for 2 min, chilled rapidly on iceprior to loading on an 8 M urea-10% polyacrylamide gel, andelectrophoresed on an ABI 373 DNA sequencer at 1400 volts. Fluorescentligation products were analyzed and parameters of an exponentialequation fit to the data using the Deltagraph Pro 3.5. software. TheX-axis indicated the different amounts of the Normal Template, while theY-axis indicated the amount of LDR product generated. (▪, Δ, □)represents 0.5 nM (10 fmol), 2.5 nM (50 fmol), and 5 nM (100 fmol),respectively of the wild type probes used with the wild-type enzymewhereas (, ♦, ∘) represents 0.5 (10 fmol), 2.5 nM (50 fmol), and 5 nM(100 fmol), of the wild type probes used with the K294R mutant enzyme.

DETAILED DESCRIPTION OF THE INVENTION

One aspect of the present invention relates to a method for detecting ina sample one or more minority target nucleotide sequences which differfrom one or more majority target nucleotide sequences by one or moresingle-base changes, insertions, deletions, or translocations, whereinthe minority target nucleotide sequences are present in the sample inlesser amounts than the majority nucleotide sequences.

One or more oligonucleotide probe sets are provided for use inconjunction with this method. Each set includes (a) a firstoligonucleotide probe having a target-specific portion and (b) a secondoligonucleotide probe having a target-specific portion. Theoligonucleotide probes in a particular set are suitable for ligationtogether when hybridized adjacent to one another on a correspondingtarget nucleotide sequence, but have a mismatch which interferes withsuch ligation when hybridized to any other nucleotide sequence presentin the sample.

The sample, the one or more oligonucleotide probe sets, and a ligase areblended to form a ligase detection reaction mixture. The ligasedetection reaction mixture is subjected to one or more ligase detectionreaction cycles comprising a denaturation treatment and a hybridizationtreatment. In the denaturation treatment, any hybridizedoligonucleotides are separated from the target nucleotide sequences. Thehybridization treatment involves hybridizing the oligonucleotide probesets at adjacent positions in a base-specific manner to the respectivetarget nucleotide sequences, if present in the sample. The hybridizedoligonucleotide probes from each set ligate to one another to form aligation product sequence containing the target-specific portionsconnected together. The ligation product sequence for each set isdistinguishable from other nucleic acids in the ligase detectionreaction mixture. The oligonucleotide probe sets may hybridize toadjacent sequences in the sample other than the respective targetnucleotide sequences but do not ligate together due to the presence ofone or more mismatches. When hydridized oligonucleotide probes do notligate, they individually separate during the denaturation treatment.

After the ligase detection reaction mixture is subjected to one or moreligase detection reaction cycles, ligation product sequences aredetected. As a result, the presence of the minority target nucleotidesequence in the sample can be identified.

In effecting detection/quantification, there are 3 techniques ofpracticing the PCR/LDR process in accordance with the present inventionwith each being practiced using either of two formats. Moreparticularly, the LDR phase can be carried out by (1) excludingwild-type allele-specific oligonucleotide probes from the LDR phase toavoid overwhelming signal from the minority mutant target and adding nomarker, (2) excluding wild-type allele-specific oligonucleotide probesfrom the LDR phase but adding a marker to that phase, and (3) utilizingwild-type allele-specific oligonucleotide probes in the LDR phase at lowlevels and/or modified forms of those probes to yield less ligationproduct corresponding to the majority target which prevents signal fromthe minority mutant target from being overwhelmed. One detection formatalternative involves use of capillary electrophoresis or gelelectrophoresis and a fluorescent quantification procedure.Alternatively, detection can be carried out by capture on an array ofcapture oligonucleotide addresses and fluorescent quantification. Thesealternatives are explained more fully with reference to FIGS. 1-9.

FIG. 1 depicts the detection of cancer-associated mutations wherewild-type allele-specific oligonucleotide probes are excluded from theLDR phase to avoid overwhelming signal from minority mutant target andno marker is added to the LDR phase. In step 1, after DNA samplepreparation, multiple exons are subjected to PCR amplification using Taqpolymerase under hot start conditions with target-specificoligonucleotide primers. The extension products produced during PCR arethen diluted 1/20 during step 2. In step 3, the extension products aremixed with oligonucleotide probes containing allele-specific portionsand common portions and the LDR phase of the process is initiated byaddition of Taq ligase under hot start conditions. During LDR,oligonucleotide probes ligate to their adjacent oligonucleotide only inthe presence of target sequence which gives perfect complementarity atthe ligation junction. Absence of wild-type allele-specificoligonucleotide probes, and consequently absence of wild-type specificligation product prevents the ligation detection reaction signalgenerated by minority mutant target from being overwhelmed.

The products can be detected by either of two formats. In the format ofstep 4a, products are separated by capillary gel electrophoresis, andfluorescent signals are quantified. On the other hand, in the format ofstep 4b, products are detected by specific hybridization tocomplementary sequences on an addressable array.

FIG. 2 depicts the detection of cancer-associated mutations wherewild-type allele-specific oligonucleotide probes are excluded from theLDR phase to avoid overwhelming signal from minority mutant target and amarker is added to the LDR phase. In step 1, after DNA samplepreparation, multiple exons are subjected to PCR amplification using Taqpolymerase under hot start conditions with target-specificoligonucleotide primers. Fluorescent quantification of PCR products canbe achieved using capillary or gel electrophoresis in step 2. In step 3,the products are spiked with a 1/100 dilution of marker DNA (for each ofthe fragments). This DNA is homologous to wild type DNA, except itcontains a mutation which is not observed in cancer cells, but which maybe readily detected with the appropriate LDR probes. In step 4, themixed DNA products from the PCR phase are then diluted 20-fold intofresh LDR buffer containing LDR oligonucleotide probes containingallele-specific portions and common portions. Step 5 involves the LDRphase of the process which is initiated by addition of Taq ligase underhot start conditions. During LDR, oligonucleotide probes ligate to theiradjacent oligonucleotide probes only in the presence of target sequencewhich gives perfect complementarity at the junction site.

The products may be detected in either of two formats. In the format ofstep 6a, products are separated by capillary or gel electrophoresis, andfluorescent signals are quantified. Ratios of mutant peaks to markerpeaks give the approximate amount of cancer-associated mutations presentin the original sample divided by 100. In the format of step 6b,products are detected by specific hybridization to complementarysequences on an addressable array. Ratios of fluorescent signals inmutant dots to marker dots give the approximate amount of cancermutations present in the original sample divided by 100.

FIG. 3 depicts the detection of additional cancer-associated mutationswhere additional wild-type allele-specific oligonucleotide probes areutilized in the LDR phase at low levels and/or are modified to yieldless ligation product corresponding to the majority target. In step 1,after DNA sample preparation, multiple exons are subjected to PCRamplification using Taq polymerase under hot start conditions withtarget-specific oligonucleotide primers. The extension products producedduring PCR are then diluted 1/20 during step 2. In step 3, the extensionproducts are mixed with oligonucleotide probes containingallele-specific portions and common portions and the LDR phase of theprocess is initiated by addition of Taq ligase under hot startconditions. During LDR, oligonucleotide probes ligate to their adjacentoligonucleotide only in the presence of target sequence which givesperfect complementarity at the ligation junction. Due to theconcentration and/or modification of the wild-type allele-specificoligonucleotide probes, the level of ligation product generated withthese probes is comparable to the amount of ligation product generatedfrom the minority target nucleotide sequences.

The products can be detected by either of two formats. In the format ofstep 4a, products are separated by capillary or gel electrophoresis, andfluorescent signal quantified. By way of example, consider the low leveland/or modified wild-type allele-specific oligonucleotide probesligating on a given amount of majority target nucleotide sequence (i.e.1 picomole) generating the same amount of ligation product as generatedfrom a given minority target sequence (using minority allele-specificoligonucleotide probes) present as a 100-fold dilution (i.e. 10femtomoles) in the same amount (i.e. 1 picomole) of majority targetnucleotide sequence. The ratio of mutant peaks to wild-type peaks givesthe approximate amount of minority target (cancer-associated mutations)present in the original sample divided by 100. In the format of step 4b,products are detected by specific hybridization to complementarysequences on an addressable array. Amount of minority product isquantified as described above.

The ligase detection reaction is described generally in WO 90/17239 toBarany et al., F. Barany et al., “Cloning, Overexpression and NucleotideSequence of a Thermostable DNA Ligase-Encoding Gene,” Gene, 109:1-11(1991), and F. Barany, “Genetic Disease Detection and DNA AmplificationUsing Cloned Thermostable Ligase,” Proc. Natl. Acad. Sci. USA,88:189-193 (1991), the disclosures of which are hereby incorporated byreference. In accordance with the present invention, the ligasedetection reaction can use 2 sets of complementary oligonucleotides.This is known as the ligase chain reaction which is described in the 3immediately preceding references, which are hereby incorporated byreference. Alternatively, the ligase detection reaction can involve asingle cycle which is known as the oligonucleotide ligation assay. SeeLandegren, et al., “A Ligase-Mediated Gene Detection Technique,” Science241:1077-80 (1988); Landegren, et al., “DNA Diagnostics—MolecularTechniques and Automation,” Science 242:229-37 (1988); and U.S. Pat. No.4,988,617 to Landegren, et al., which are hereby incorporated byreference.

During the ligase detection reaction phase, the denaturation treatmentis carried out at a temperature of 80-105 C., while hybridization takesplace at 50-85 C. Each cycle comprises a denaturation treatment and athermal hybridization treatment which in total is from about one to fiveminutes long. Typically, the ligation detection reaction involvesrepeatedly denaturing and hybridizing for 2 to 50 cycles. The total timefor the ligase detection reaction phase is 1 to 250 minutes.

The oligonucleotide probe sets can be in the form of ribonucleotides,deoxynucleotides, modified ribonucleotides, modifieddeoxyribonucleotides, modified phosphate-sugar-backbone oligonucleotides(described infra), nucleotide analogs, and mixtures thereof.

In one variation, the oligonucleotides of the oligonucleotide probe setseach have a hybridization or melting temperature (i.e. T_(m)) of 66-70C. These oligonucleotides are 20-28 nucleotides long.

The oligonucleotide probe sets, as noted above, have a reporter labelsuitable for detection. Useful labels include chromophores, fluorescentmoieties, enzymes, antigens, heavy metals, magnetic probes, dyes,phosphorescent groups, radioactive materials, chemiluminescent moieties,and electrochemical detecting moieties.

The polymerase chain reaction process is fully described in H. Erlich,et. al., “Recent Advances in the Polymerase Chain Reaction,” Science252: 1643-50 (1991); M. Innis, et. al., PCR Protocols: A Guide toMethods and Applications, Academic Press: New York (1990); and R. Saiki,et. al., “Primer-directed Enzymatic Amplification of DNA with aThermostable DNA Polymerase,” Science 239: 487-91 (1988), which arehereby incorporated by reference.

A particularly important aspect of the present invention is itscapability to quantify the amount of target nucleotide sequence in asample. This can be achieved in a number of ways by establishingstandards which can be internal (i.e. where the standard establishingmaterial is amplified and detected with the sample) or external (i.e.where the standard establishing material is not amplified, and isdetected with the sample).

In accordance with one quantification method, the signal generated byligation product sequences produced from the sample being analyzed, aredetected. The strength of this signal is compared to a calibration curveproduced from signals generated by ligation product sequences in sampleswith known amounts of target nucleotide sequence. As a result, theamount of target nucleotide sequence in the sample being analyzed can bedetermined. This technique involves use of an external standard.

Another quantification method, in accordance with the present invention,relates to an internal standard. Here, a known amount of one or moremarker target nucleotide sequences is added to the sample. In addition,one or a plurality of marker-specific oligonucleotide probe sets areadded along with the ligase, the previously-discussed oligonucleotideprobe sets, and the sample to a mixture. The marker-specificoligonucleotide probe sets have (1) a first oligonucleotide probe with atarget-specific portion complementary to the marker target nucleotidesequence, and (2) a second oligonucleotide probe with a target-specificportion complementary to the marker target nucleotide sequence and adetectable reporter label. The oligonucleotide probes in a particularmarker-specific oligonucleotide set are suitable for ligation togetherwhen hybridized adjacent to one another on a corresponding marker targetnucleotide sequence. However, there is a mismatch which interferes withsuch ligation when hybridized to any other nucleotide sequence presentin the sample or added marker sequences. The presence of ligationproduct sequences is identified by detection of reporter labels. Theamount of target nucleotide sequences in the sample is then determinedby comparing the amount of ligation product sequence generated fromknown amounts of marker target nucleotide sequences with the amount ofother ligation product sequences.

Another quantification method, in accordance with the present invention,involves analysis of a sample containing two or more of a plurality oftarget nucleotide sequences with a plurality of sequence differences.Here, ligation product sequences corresponding to the target nucleotidesequences are detected and distinguished by any of thepreviously-discussed techniques. The relative amounts of the targetnucleotide sequences in the sample are then quantified by comparing therelative amounts of ligation product sequences generated. This providesa quantitative measure of the relative level of the target nucleotidesequences in the sample.

Another quantification method, in accordance with the present invention,involves analysis of a sample containing two or more of a plurality oftarget nucleotide sequences with a plurality of sequence differences,where one or more target nucleotide sequences is in excess (majority)over other minority target nucleotide sequences. Here, in addition tothe allele-specific oligonucleotide probes for the minority targetnucleotide sequences, modified wild-type allele-specific oligonucleotideprobes are also utilized in the LDR phase at low levels and/or aremodified to yield less ligation product corresponding to the majoritytarget. The presence of both minority target specific ligation productsand majority target specific ligation products is identified bydetection of reporter labels. The amount of minority target nucleotidesequences in the sample is determined by comparing the amount of lowyield ligation product sequences generated from the majority targetnucleotide sequences with the amount of other ligation products.

The preferred thermostable ligase is that derived from Thermusaquaticus. This enzyme can be isolated from that organism. M. Takahashi,et al., “Thermophillic DNA Ligase,” J. Biol. Chem. 259:1004147 (1984),which is hereby incorporated by reference. Alternatively, it can beprepared recombinantly. Procedures for such isolation as well as therecombinant production of Thermus aquaticus ligase (as well as Thermusthemophilus ligase) are disclosed in WO 90/17239 to Barany, et. al., andF. Barany, et al., “Cloning, Overexpression and Nucleotide Sequence of aThermostable DNA-Ligase Encoding Gene,” Gene 109:1-11 (1991), which arehereby incorporated by reference. These references contain completesequence information for this ligase as well as the encoding DNA. Othersuitable ligases include E. coli ligase, T4 ligase, and Pyrococcusligase.

The ligation detection reaction mixture may include a carrier DNA, suchas salmon sperm DNA.

The hybridization step in the ligase detection reaction, which ispreferably a thermal hybridization treatment discriminates betweennucleotide sequences based on a distinguishing nucleotide at theligation junctions. The difference between the target nucleotidesequences can be, for example, a single nucleic acid base difference, anucleic acid deletion, a nucleic acid insertion, or rearrangement. Suchsequence differences involving more than one base can also be detected.Preferably, the oligonucleotide probe sets have substantially the samelength so that they hybridize to target nucleotide sequences atsubstantially similar hybridization conditions. As a result, the processof the present invention is able to detect infectious diseases, geneticdiseases, and cancer. It is also useful in environmental monitoring,forensics, and food science.

A wide variety of infectious diseases can be detected by the process ofthe present invention. Typically, these are caused by bacterial, viral,parasite, and fungal infectious agents. The resistance of variousinfectious agents to drugs can also be determined using the presentinvention.

Bacterial infectious agents which can be detected by the presentinvention include Escherichia coli, Salmonella, Shigella, Klebsiella,Pseudomonas, Listeria monocytogenes, Mycobacterium tuberculosis,Mycobacterium avium-intracellulare, Yersinia, Francisella, Pasteurella,Brucella, Clostridia, Bordetella pertussis, Bacteroides, Staphylococcusaureus, Streptococcus pneumonia, B-Hemolytic strep., Corynebacteria,Legionella, Mycoplasma, Ureaplasma, Chlamydia, Neisseria gonorrhea,Neisseria meningitides, Hemophilus influenza, Enterococcus faecalis,Proteus vulgaris, Proteus mirabilis, Helicobacter pylori, Treponemapalladium, Borrelia burgdorferi, Borrelia recurrentis, Rickettsialpathogens, Nocardia, and Acitnomycetes.

Fungal infectious agents which can be detected by the present inventioninclude Cryptococcus neoformans, Blastomyces dermatitidis, Histoplasmacapsulatum, Coccidioides immitis, Paracoccidioides brasiliensis, Candidaalbicans, Aspergillus fumigautus, Phycomycetes (Rhizopus), Sporothrixschenckii, Chromomycosis, and Maduromycosis.

Viral infectious agents which can be detected by the present inventioninclude human immunodeficiency virus, human T-cell lymphocytotrophicvirus, hepatitis viruses (e.g., Hepatitis B Virus and Hepatitis CVirus), Epstein-Barr Virus, cytomegalovirus, human papillomaviruses,orthomyxo viruses, paramyxo viruses, adenoviruses, corona viruses,rhabdo viruses, polio viruses, toga viruses, bunya viruses, arenaviruses, rubella viruses, and reo viruses.

Parasitic agents which can be detected by the present invention includePlasmodium falciparum, Plasmodium malaria, Plasmodium vivax, Plasmodiumovale, Onchoverva volvulus, Leishmania, Trypanosoma spp., Schistosomaspp., Entamoeba histolytica, Cryptosporidum, Giardia spp., Trichimonasspp., Balatidium coli, Wuuchereria bancrofti, Toxoplasma spp.,Enterobius vermicularis, Ascaris lumbricoides, Trichuris trichiura,Dracunculus medinesis, trematodes, Diphyllobothrium latum, Taenia spp.,Pneumocystis carinii, and Necator americanis.

The present invention is also useful for detection of drug resistance byinfectious agents. For example, vancomycin-resistant Enterococcusfaecium, methicillin-resistant Staphylococcus aureus,penicillin-resistant Streptococcus pneumoniae, multi-drug resistantMycobacterium tuberculosis, and AZT-resistant human immunodeficiencyvirus can all be identified with the present invention.

Genetic diseases can also be detected by the process of the presentinvention. This can be carried out by prenatal or post-natal screeningfor chromosomal and genetic aberrations or for genetic diseases.Examples of detectable genetic diseases include: 21 hydroxylasedeficiency, cystic fibrosis, Fragile X Syndrome, Turner Syndrome,Duchenne Muscular Dystrophy, Down Syndrome or other trisomies, heartdisease, single gene diseases, HLA typing, phenylketonuria, sickle cellanemia, Tay-Sachs Disease, thalassemia, Klinefelter Syndrome, HuntingtonDisease, autoimmune diseases, lipidosis, obesity defects, hemophilia,inborn errors of metabolism, and diabetes.

Cancers which can be detected by the process of the present inventiongenerally involve oncogenes, tumor suppressor genes, or genes involvedin DNA amplification, replication, recombination, or repair. Examples ofthese include: BRCA1 gene, p53 gene, APC gene, Her2/Neu amplification,Bcr/Ab1, K-ras gene, and human papillomavirus Types 16 and 18. Variousaspects of the present invention can be used to identify amplifications,large deletions as well as point mutations and smalldeletions/insertions of the above genes in the following common humancancers: leukemia, colon cancer, breast cancer, lung cancer, prostatecancer, brain tumors, central nervous system tumors, bladder tumors,melanomas, liver cancer, osteosarcoma and other bone cancers, testicularand ovarian carcinomas, head and neck tumors, and cervical neoplasms.

In the area of environmental monitoring, the present invention can beused for detection, identification, and monitoring of pathogenic andindigenous microorganisms in natural and engineered ecosystems andmicrocosms such as in municipal waste water purification systems andwater reservoirs or in polluted areas undergoing bioremediation. It isalso possible to detect plasmids containing genes that can metabolizexenobiotics, to monitor specific target microorganisms in populationdynamic studies, or either to detect, identify, or monitor geneticallymodified microorganisms in the environment and in industrial plants.

The present invention can also be used in a variety of forensic areas,including for human identification for military personnel and criminalinvestigation, paternity testing and family relation analysis, HLAcompatibility typing, and screening blood, sperm, or transplantationorgans for contamination.

In the food and feed industry, the present invention has a wide varietyof applications. For example, it can be used for identification andcharacterization of production organisms such as yeast for production ofbeer, wine, cheese, yogurt, bread, etc. Another area of use is withregard to quality control and certification of products and processes(e.g., livestock, pasteurization, and meat processing) for contaminants.Other uses include the characterization of plants, bulbs, and seeds forbreeding purposes, identification of the presence of plant-specificpathogens, and detection and identification of veterinary infections.

Desirably, the oligonucleotide probes are suitable for ligation togetherat a ligation junction when hybridized adjacent to one another on acorresponding target nucleotide sequence due to perfect complementarityat the ligation junction. However, when the oligonucleotide probes inthe set are hybridized to any other nucleotide sequence present in thesample, there is a mismatch at a base at the ligation junction whichinterferes with ligation. Most preferably, the mismatch is at the baseat the 3′ base at the ligation junction. Alternatively, the mismatch canbe at the bases adjacent to bases at the ligation junction.

As noted supra, detection and quantification can be carried out usingcapillary or gel electrophoresis or on a solid support with an arraycapture oligonucleotides.

FIG. 4 is a schematic diagram depicting the PCR/LDR process of FIG. 1using electrophoresis to separate ligation products. More particularly,this diagram relates to detection of codon 12 of the K-ras gene whichhas a GGT sequence that codes for glycine (“Gly”). A small percentage ofthe cells contain the G to A mutation in GAT, which codes for asparticacid (“Asp”). As illustrated, this process involves an initial PCRamplification in step 1, an LDR procedure in step 2, and a separation offluorescent products followed by quantification in step 3.Alternatively, step 3 can involve ethidium bromide staining or runningan additional LDR reaction on diluted product using normaloligonucleotide probes (See FIGS. 23 and 29 infra). The LDR probes forwild-type (i.e. normal) sequences are missing from the reaction. If thenormal LDR probes (with the discriminating base being G) were included,they would ligate to the common probes and overwhelm any signal comingfrom the minority mutant target. Instead, as shown in FIG. 4, theexistence of a 44 base ligation product sequence with fluorescent labelF1 coupled to a single nucleotide (designated N₁) and the A_(n) tailindicates the presence of the aspartic acid encoding mutant. Thisligation product sequence has the same F1 label as that formed by theexistence of the arginine and valine encoded mutations. However, thesesequences are distinguishable by virtue of their different lengths dueto different length tails and different numbers of nucleotides Ncoupling the label to the remainder of the ligation product sequence.More particularly, the presence of an F1 labelled, 48 base ligationproduct sequence suggests the presence of the arginine encoding codon,while the presence of an F1 labelled, 46 base ligation product sequenceindicates the presence of the valine encoding codon. These ligationproduct sequences are distinguished by size with the longer productshaving a lower electrophoretic mobility. The F2 labelled ligationproducts are similarly distinguished by their length which varies as aresult of the different length tails and the number of nucleotides Ncoupling the label to the remainder of the ligation product sequence.More particularly, the 49 base ligation product sequence (due to 2nucleotides N coupling the label and the A_(n+4) tail) indicates thepresence of the cysteine encoding codon, the 47 base ligation productsequence (due to no nucleotides N coupling the label and the A_(n+4)tail) indicates the presence of the serine encoding codon, and the 45base ligation product sequence (due to 2 nucleotides N coupling thelabel and the A_(n) tail) indicates the presence of the alanine encodingcodon.

FIG. 5 is a schematic diagram depicting the PCR/LDR process of FIG. 2using electrophoresis to separate ligation products. More particularly,this diagram relates to detection of codon 12 of the K-ras gene whichhas a GGT sequence that codes for glycine (“Gly”). A small percentage ofthe cells contain the G to A mutation in GAT, which codes for asparticacid (“Asp”). As illustrated, this process involves an initial PCRamplification in step 1, an LDR procedure in step 2, and a separation offluorescent products followed by quantification in step 3. Afteramplification, the PCR products are quantified. A marker template isadded prior to the LDR phase where both allele-specific andmarker-specific oligonucleotide probes are utilized. The LDR probes forwild-type (i.e. normal) sequences are missing from the reaction. If thenormal LDR probes (with the discriminating base being G) were included,they would ligate to the common probes and overwhelm any signal comingfrom the minority mutant target. Instead, as shown in FIG. 5, theexistence of a 44 base ligation product sequence with fluorescent labelF1 coupled to a single nucleotide (designated N₁) and the A_(n) tailindicates the presence of the aspartic acid encoding mutant. Thisligation product sequence has the same F1 label as that formed by theexistence of the arginine and valine encoded mutations. However, thesesequences are distinguishable by virtue of their different lengths dueto different length tails and different numbers of nucleotides Ncoupling the label to the remainder of the ligation product sequence.More particularly, the presence of an F1 labelled, 48 base ligationproduct sequence suggests the presence of the arginine encoding codon,while the presence of an F1 labelled, 46 base ligation product sequenceindicates the presence of the valine encoding codon. These ligationproduct sequences are distinguished by size with the longer productshaving a lower electrophoretic mobility. The F2 labelled ligationproducts are similarly distinguished by their length which varies as aresult of the different length tails and the number of nucleotides Ncoupling the label to the remainder of the ligation product sequence.More particularly, the 49 base ligation product sequence (due to 2nucleotides N coupling the label and the A_(n+4) tail) indicates thepresence of the cysteine encoding codon, the 47 base ligation productsequence (due to no nucleotides N coupling the label and the A_(n+4)tail) indicates the presence of the serine encoding codon, and the 45base ligation product sequence (due to 2 nucleotides N coupling thelabel and the A_(n) tail) indicates the presence of the alanine encodingcodon. The ligation product formed by the marker-specificoligonucleotide probe is 43 bases and has the F₂ label (due to 0nucleotides N coupling the label and the A_(n) tail). As discussedabove, the amount of minority target nucleotide sequences in the sampleis determined by comparing the amount of ligation product sequencegenerated from known amounts of marker target nucleotide sequences withthe amount of other ligation product sequences.

FIG. 6 is a schematic diagram depicting the PCR/LDR process of FIG. 3using electrophoresis to separate ligation products. More particularly,this diagram relates to detection of codon 12 of the K-ras gene whichhas a GGT sequence that codes for glycine (“Gly”). A small percentage ofthe cells contain the G to A mutation in GAT, which codes for asparticacid (“Asp”). As illustrated, this process involves an initial PCRamplification in step 1, an LDR procedure in step 2, and a separation offluorescent products followed by quantification in step 3. The LDRprobes for wild-type (i.e. normal) sequences are used at low leveland/or are modified to yield less ligation product sequencecorresponding to wild type target nucleotide sequence. As shown in FIG.6, the existence of a 44 base ligation product sequence with fluorescentlabel F1 coupled to a single nucleotide (designated N₁) and the A_(n)tail indicates the presence of the aspartic acid encoding mutant. Thisligation product sequence has the same F1 label as that formed by theexistence of the arginine and valine encoded mutations. However, thesesequences are distinguishable by virtue of their different lengths dueto different length tails and different numbers of nucleotides Ncoupling the label to the remainder of the ligation product sequence.More particularly, the presence of an F1 labelled, 48 base ligationproduct sequence suggests the presence of the arginine encoding codon,while the presence of an F1 labelled, 46 base ligation product sequenceindicates the presence of the valine encoding codon. These ligationproduct sequences are distinguished by size with the longer productshaving a lower electrophoretic mobility. The F2 labelled ligationproducts are similarly distinguished by their length which varies as aresult of the different length tails and the number of nucleotides Ncoupling the label to the remainder of the ligation product sequence.More particularly, the 49 base ligation product sequence (due to 2nucleotides N coupling the label and the A_(n+4) tail) indicates thepresence of the cysteine encoding codon, the 47 base ligation productsequence (due to no nucleotides N coupling the label and the A_(n+4)tail) indicates the presence of the serine encoding codon, and the 45base ligation product sequence (due to 2 nucleotides N coupling thelabel and the A_(n) tail) indicates the presence of the alanine encodingcodon. The ligation product formed by the wild type allele-specificoligonucleotide probe is 43 bases and has the F₂ label (due to 0nucleotides N coupling the label and the A_(n) tail). In the labelledprobe forming that ligation product, there is a base N located 3 basepositions away from the ligation junction which can be either theconventional, proper nucleotide for the wild type target (if that probeis used at low level), or a mismatch, or a nucleotide base analogue. Useof a mismatched nucleotide, a nucleotide base analogue, and/or amodification in the sugar phosphate backbone reduces the amount ofligation product formed off wild-type target. Thus, the presence of wildtype target can be detected without overwhelming the signal generated bythe presence of minority mutant target. The amount of minority targetnucleotide sequences in the sample is determined by comparing the amountof low yield ligation product sequences generated from the majoritytarget nucleotide sequences with the amount of other ligation products.

FIGS. 4-6 show the use of the ligase detection reaction to detectmismatches at the 3′ end of the distinguishing oligonucleotide probe. Inother cases, however, the mismatch can be at the penultimate position tothe 3′ end or and the third position away from the 3′ end.

The use of capillary and gel electrophoresis for such purposes is wellknown. See e.g., Grossman, et. al., “High-density Multiplex Detection ofNucleic Acid Sequences: Oligonucleotide Ligation Assay andSequence-coded Separation,” Nucl. Acids Res. 22(21): 4527-34 (1994),which is hereby incorporated by reference.

FIG. 7 is a schematic diagram depicting the PCR/LDR process of FIG. 1for detection of cancer-associated mutations at adjacent alleles usingan addressable array. FIG. 7 relates to the detection of codon 12 of theK-ras gene which has a wild-type GGT sequence that codes for glycine(“Gly”) and minority mutant GAT sequence coding for aspartic acid(“Asp”). The process of FIG. 7 involves an initial PCR amplification instep 1, an LDR procedure in step 2, and capture on a solid support instep 3. As in FIG. 4, the LDR probes for the wild-type target sequenceare missing from the reaction to avoid overwhelming signal produced bythe mutant target sequence. According to this embodiment of the presentinvention, as shown in FIG. 7, the presence of the aspartic acidencoding GAT sequence produces a ligation product sequence with label Fand addressable array-specific portion Z4. The existence of such aligation product sequence is indicated by the presence of a nucleicacid, having label F, hybridized at an address on a solid support with acapture oligonucleotide complementary to addressable array-specificportion Z4. As shown in FIG. 7, the support has an array of addresseswith capture oligonucleotides complementary to different addressablearray-specific portions Z1 to Z6. Since common oligonucleotide probeswith label F are used, by observing which site on the solid support theyhybridize to, different ligation product sequences are distinguished.

FIG. 8 is a schematic diagram depicting the PCR/LDR process of FIG. 2for detection of cancer-associated mutations at adjacent alleles usingan addressable array. FIG. 8 relates to the detection of codon 12 of theK-ras gene which has a wild-type GGT sequence that codes for glycine(“Gly”) and minority mutant GAT sequence coding for aspartic acid(“Asp”). The process of FIG. 8 involves an initial PCR amplification instep 1, an LDR procedure in step 2, and capture on a solid support instep 3. As in FIG. 5, the LDR probes for the wild-type target sequenceare missing from the reaction to avoid overwhelming signal produced bythe mutant target sequence. According to this embodiment of the presentinvention, as shown in FIG. 8, the presence of the aspartic acidencoding GAT sequence produces a ligation product sequence with label Fand addressable array-specific portion Z4. The existence of such aligation product sequence is indicated by the presence of a nucleicacid, having label F, hybridized at an address on a solid support with acapture oligonucleotide complementary to addressable array-specificportion Z4. As shown in FIG. 8, the support has an array of addresseswith capture oligonucleotides complementary to different addressablearray-specific portions Z1 to Z7. Since common oligonucleotide probeswith label F are used, by observing which site on the solid support theyhybridize to, different ligation product sequences are distinguished.The presence of ligation product sequence produced from amarker-specific probe is indicated by the existence of a nucleic acid,having label F, hybridized at an address on a solid support with acapture oligonucleotide complementary to addressable array-specificportion Z7. The amount of target nucleotide sequences in the sample isdetermined by comparing the amount of ligation product sequencegenerated from known amounts of marker target nucleotide sequences withthe amount of other ligation product sequences.

FIG. 9 is a schematic diagram depicting the PCR/LDR process of FIG. 3for detection of cancer-associated mutations at adjacent alleles usingan addressable array. FIG. 9 relates to the detection of codon 12 of theK-ras gene which has a wild-type GGGT sequence that codes for glycine(“Gly”) and minority mutant GAT sequence coding for aspartic acid(“Asp”). The process of FIG. 9 involves an initial PCR amplification instep 1, an LDR procedure in step 2, and capture on a solid support instep 3. The LDR probes for wild-type (i.e. normal) sequences are used atlow level and/or are modified to yield less ligation product sequencecorresponding to wild type target nucleotide sequence. According to thisembodiment of the present invention, as shown in FIG. 9, the presence ofthe aspartic acid encoding GAT sequence produces a ligation productsequence with label F and addressable array-specific portion Z4. Theexistence of such a ligation product sequence is indicated by thepresence of a nucleic acid, having label F, hybridized at an address ona solid support with a capture oligonucleotide complementary toaddressable array-specific portion Z4. As shown in FIG. 9, the supporthas an array of addresses with capture oligonucleotides complementary todifferent addressable array-specific portions Z1 to Z7. Since commonoligonucleotide probes with label F are used, different ligation productsequences are distinguished by which site on the solid support theyhybridize to. The ligation product formed by the wild typeallele-specific oligonucleotide probe is indicated by the existence of anucleic acid, having label F, hybridized at an address on a solidsupport with a capture oligonucleotide complementary to addressablearray-specific portion Z7. In the labelled probe forming that ligationproduct, there is a base N located 3 base positions away from theligation junction that can be either a conventional nucleotide for thewild type target (if that probe is used at low level), or a mismatchnucleotide, or a nucleotide base analogue. Use of a mismatchednucleotide, a nucleotide base analogue, and/or a modification in thesugar phosphate backbone reduces the amount of ligation product formedoff wild-type target. Thus, the presence of wild type target can bedetected without overwhelming the signal generated by the presence ofminority mutant target. The amount of minority target nucleotidesequences in the sample is determined by comparing the amount of lowyield ligation product sequences generated from the majority targetnucleotide sequences with the amount of other ligation products.

The use of a solid support with an array of capture oligonucleotides isfully disclosed in pending provisional U.S. patent application Ser. No.60/011,359, which is hereby incorporated by reference. When using sucharrays, the oligonucleotide probes used in the above-described LDR phasehave an addressable array-specific portion. After the LDR phase iscompleted, the addressable array-specific portions for the products ofsuch processes remain single stranded and are caused to hybridize to thecapture oligonucleotides during a capture phase. See Newton, et al.,“The Production of PCR Products With 5′ Single-Stranded Tails UsingPrimers That Incorporate Novel Phosphoramidite Intermediates,” Nucl.Acids Res. 21(5):1155-62 (1993), which is hereby incorporated byreference.

During the capture phase of the process, the mixture is contacted withthe solid support at a temperature of 45-90 C. and for a time period ofup to 60 minutes. Hybridizations may be accelerated by adding cations,volume exclusion or chaotropic agents. When an array consists of dozensto hundreds of addresses, it is important that the correct ligationproduct sequences have an opportunity to hybridize to the appropriateaddress. This may be achieved by the thermal motion of oligonucleotidesat the high temperatures used, by mechanical movement of the fluid incontact with the array surface, or by moving the oligonucleotides acrossthe array by electric fields. After hybridization, the array is washedsequentially with a low stringency wash buffer and then a highstringency wash buffer.

It is important to select capture oligonucleotides and addressablenucleotide sequences which will hybridize in a stable fashion. Thisrequires that the oligonucleotide sets and the capture oligonucleotidesbe configured so that the oligonucleotide sets hybridize to the targetnucleotide sequences at a temperature less than that which the captureoligonucleotides hybridize to the addressable array-specific portions.Unless the oligonucleotides are designed in this fashion, false positivesignals may result due to capture of adjacent unreacted oligonucleotidesfrom the same oligonucleotide set which are hybridized to the target.

The capture oligonucleotides can be in the form of ribonucleotides,deoxyribonucleotides, modified ribonucleotides, modifieddeoxyribonucleotides, peptide nucleotide analogues, modified peptidenucleotide analogues, modified phosphate-sugar backboneoligonucleotides, nucleotide analogues, and mixtures thereof.

Where an array is utilized, the detection phase of the process involvesscanning and identifying if LDR products have been produced andcorrelating the presence of such products to a presence or absence ofthe target nucleotide sequence in the test sample. Scanning can becarried out by scanning electron microscopy, confocal microscopy,charge-coupled device, scanning tunneling electron microscopy, infraredmicroscopy, atomic force microscopy, electrical conductance, andfluorescent or phosphor imaging. Correlating is carried out with acomputer.

The present invention is useful in distinguishing a minority targetnucleotide sequence from the majority nucleotide sequence in a sample ata respective ratio of 1:500 for a G:T or T:G mismatch between themajority target nucleotide sequence and one of the oligonucleotideprobes. Further, this method can distinguish a minority targetnucleotide sequence from the majority nucleotide sequence in a sample ata respective ratio of 1:2000 for other than a G:T or T:G mismatchbetween the majority target nucleotide sequence and one of theoligonucleotide probes.

For low abundance multiple allele differences at multiple nearby oradjacent positions, the process of the present invention distinguishesminority target nucleotide sequences from the majority target sequenceat a respective ratio of 1:100 for all mismatches between the majoritytarget nucleotide sequence and one of the oligonucleotide probes. Insuch situations, the minority target nucleotide sequence to majoritytarget nucleotide sequence respective ratio is 1:500 for other than G:Tor T:G mismatches between the majority target nucleotide sequence andone of the oligonucleotide probes.

The second aspect of the present invention also relates to a method foridentifying one or more of a plurality of sequences differing by one ormore single-base changes, insertions, deletions, or translocations in aplurality of target nucleotide sequences. As noted above, a sample andone or more oligonucleotide probe sets are blended with a ligase to forma ligase detection reaction mixture. The ligase detection reactionmixture is subjected to one or more ligase detection reaction cycles,and the presence of ligation product sequences is detected. Here,however, a thermostable mutant ligase is utilized. This ligase ischaracterized by a fidelity ratio which is defined as the initial rateconstant for ligating the first and second oligonucleotide probeshybridized to a target nucleotide sequence with a perfect match at theligation junction between the target nucleotide sequence and theoligonucleotide probe having its 3′ end at the ligation junction to theinitial rate constant for ligating the first and second oligonucleotideprobes hybridized to a target with a mismatch at the ligation junctionbetween the target nucleotide sequence and the oligonucleotide probehaving its 3′ end at the ligation junction. The fidelity ratio for thethermostable mutant ligase is greater than the fidelity ratio forwild-type ligase.

The use of a mutant ligase in accordance with the process of the presentinvention can be explained as follows. The specificity of an enzymaticreaction is determined by the catalytic constant, k_(cat), and theapparent binding constant, K_(M), and expressed as the specificityconstant k_(cat)/K_(M). Any modifications made on the enzyme itself,substrate, or reaction conditions, which affect k_(cat) or K_(M) orboth, will change the specificity. The use of a mutant enzyme mayinfluence the stability of the perfect matched and mismatched enzyme-DNAcomplexes to a different extent, so that discrete K_(M) effects areexerted on these ligation reactions. In a competitive reaction, such asligation of perfectly matched and mismatched substrates, the ratio ofthe specificity constant may be altered as a consequence of K_(M), andpossible k_(cat) changes for each substrate. All mutant enzymes whichsatisfy the equation below (shown for K294R) will give increaseddiscrimination of cancer-associated mutations in the presence of anexcess of normal DNA.$\frac{\left\lbrack {k_{cat}/K_{M}} \right\rbrack_{{K294R},{match}}}{\left\lbrack {k_{cat}/K_{M}} \right\rbrack_{{K294R},{mismatch}}} > \frac{\left\lbrack {k_{cat}/K_{M}} \right\rbrack_{{Wt},{match}}}{\left\lbrack {k_{cat}/K_{M}} \right\rbrack_{{Wt},{mismatch}}}$

Alternatively, the second aspect of the present invention can beexpressed in terms of a fidelity ratio (i.e. the initial rate ofligating a substrate with a perfect match at the 3′ end divided by theinitial rate of ligating a substrate with a mismatch at the 3′ end) asfollows:${\frac{\left\lbrack k_{1} \right\rbrack_{{K294R},{match}}}{\left\lbrack k_{1} \right\rbrack_{{K294R},{mismatch}}} > \frac{\left\lbrack k_{1} \right\rbrack_{{Wt},{match}}}{\left\lbrack k_{1} \right\rbrack_{{Wt},{mismatch}}}} = {{Fidelity}\quad {ratio}}$

In the above equation, [k₁]_(match) represents the initial rate constantfor ligating the first and second oligonucleotide probes hybridized to atarget nucleotide sequence with a perfect match at the ligation junctionbetween the target nucleotide sequence and the oligonucleotide probehaving its 3′ end at the ligation junction. [k₁]_(mismatch) representsthe initial rate constant for ligating the first and secondoligonucleotide probes hybridized to a target with a mismatch at theligation junction between the target nucleotide sequence and theoligonucleotide probe having its 3′ end at the ligation junction. Forthe mutant thermostable ligase, [k₁]_(match) divided by [k₁]_(mismatch)(=fidelity ratio) is greater than the fidelity ratio for wild-typeligase. All mutant enzymes which satisfy the equation above (shown forK294R) will give increased discrimination of cancer-associated mutationsin the presence of an excess of normal DNA. This can also be stated moregenerally and in terms of a signal to noise ratio as follows:$\frac{\left\lbrack {{LDR}\quad {product}} \right\rbrack_{{minority}\quad {target}} + \left\lbrack {{LDR}\quad {product}} \right\rbrack_{{majority}\quad {target}}}{\left\lbrack {{LDR}\quad {product}} \right\rbrack_{{majority}\quad {target}}} = {{Signal}\text{-}{to}\text{-}{noise}\quad {ratio}}$

In the above equation, [LDR product]_(minority target) represents theamount of ligation product sequences produced when the first and secondoligonucleotide probes hybridize to a minority target nucleotidesequence and the oligonucleotide probe having its 3′ end at the ligationjunction. [LDR product]_(majority target) represents the amount ofligation product sequences produced when the same first and secondoligonucleotide probes hybridize to the majority target nucleotidesequence with a mismatch at the ligation junction between the majoritytarget nucleotide sequence and the oligonucleotide probe having its 3′end at the ligation junction. The ligase has a signal-to-noise ratio,for the amount of ligation product sequences produced from both theminority and majority target nucleotide sequences divided by the amountof ligation product sequences produced from the same amount of majoritytarget nucleotide sequences alone.

Both mutant and wild-type ligases have associated signal-to-noise ratiosfor detection of minority mutations, and the second aspect of thepresent invention can be expressed as the mutant ligase signal-to-noiseratio is greater than the wild-type ligase signal-to-noise ratio.$\frac{\frac{\quad {\left\lbrack {{LDR}\quad {product}} \right\rbrack_{{minority}\quad {target}} + \left\lbrack {{LDR}\quad {product}} \right\rbrack_{{majority}\quad {target}}}}{\left\lbrack {{LDR}\quad {product}} \right\rbrack_{{majority}\quad {target}}}{Mutant}\quad {ligase}}{\frac{\left\lbrack {{LDR}\quad {product}} \right\rbrack_{{minority}\quad {target}} + \left\lbrack {{LDR}\quad {product}} \right\rbrack_{{majority}\quad {target}}}{\left\lbrack {{LDR}\quad {product}} \right\rbrack_{{majority}\quad {target}}}{Wild}\text{-}{type}\quad {ligase}} > 1$

The above equation may be restated more simply as:$\frac{{Signal}\text{-}{to}\text{-}{noise}\quad {ratio}\quad {for}\quad {Mutant}\quad {ligase}}{{Signal}\text{-}{to}\text{-}{noise}\quad {ratio}\quad {for}\quad {Wild} - {type}\quad {ligase}} > 1$

For the mutant thermostable ligase, the signal-to-noise ratio is greaterthan the signal-to-noise ratio for wild-type ligase. All mutant enzymeswhich satisfy the equation above will give increased discrimination ofcancer-associated mutations in the presence of an excess of normal DNA.

The third aspect of the present invention also relates to a method foridentifying one or more of a plurality of sequences differing by one ormore single-base changes, insertions, deletions, or translocations in aplurality of target nucleotide sequences. As noted above, a sample andone or more oligonucleotide probe sets are blended with a ligase to forma ligase detection reaction mixture. The ligase detection reactionmixture is subjected to one or more ligase detection reaction cycles,and the presence of ligation product sequences is then detected. Here,however, with regard to the oligonucleotide probe sets, theoligonucleotide probe which has its 3′ end at the junction whereligation occurs has a modification. This modification differentiallyalters the ligation rate when the first and second oligonucleotideprobes hybridize to a minority target nucleotide sequence in the samplewith a perfect match at the ligation junction between the minoritytarget nucleotide sequence and the oligonucleotide probe having its 3′end at the ligation junction compared to the ligation rate when thefirst and second oligonucleotide probes hybridize to the sample'smajority target nucleotide sequence with a mismatch at the ligationjunction between the majority target nucleotide sequence and thenucleotide probe having its 3′ end at the ligation junction. Ligationwith the modified oligonucleotide probe has a signal-to-noise ratio, ofthe ligation product sequence amounts for the minority and majoritytarget nucleotide sequences to the amount of ligation product sequencesproduced from the same amount of majority target sequence alone, whichis greater than the signal-to-noise ratio for ligation using anoligonucleotide probe lacking the modification.

The use of a modified oligonucleotide probe in accordance with theprocess of the present invention can be explained as follows:

Introduction of the Q₂ or Q₁₈ analogues at the 3rd position of thediscriminating primer improves the signal to noise ratio about 2 to3-fold, thereby increasing the power of the LDR system to discriminatecancer signal from background. This assay compares the ability of ligaseto discriminate the most difficult case; a T:G mismatch from a T:Aperfect match. Q₂ or Q₁₈ analogues located three nucleotides in from the3′-end of a probe enhance local melting when present in conjunction witha mismatch at the 3′-position, while at the same time preserving helixintegrity more than a mismatch when present in conjunction with a basepair match at the 3′-end. The use of a Q₂ or Q₁₈ analogue near the 3′end of a probe may influence the stability of the perfect matched andmismatched enzyme-DNA complexes to a different extent, so that discreteK_(M) effects are exerted on these ligation reactions. In a competitivereaction, such as ligation of perfectly matched and mismatchedsubstrates, the ratio of the specificity constant may be altered as aconsequence of K_(M), and possible kcat changes for each substrate. Allmodified probes which satisfy the equation below (shown for Q analogues)will give increased discrimination of cancer-associated mutations in thepresence of an excess of normal DNA.$\frac{\left\lbrack {k_{cat}/K_{M}} \right\rbrack_{{{SLP3}^{\prime}{QTT}},{match}}}{\left\lbrack {k_{cat}/K_{M}} \right\rbrack_{{{SLP3}^{\prime}{QTT}},{mismatch}}} > \frac{\left\lbrack {k_{cat}/K_{M}} \right\rbrack_{{{SLP3}^{\prime}{TTT}},{match}}}{\left\lbrack {k_{cat}/K_{M}} \right\rbrack_{{{SLP3}^{\prime}{TTT}},{mismatch}}}$

Alternatively, the third aspect of the present invention can be jexpressed in terms of a fidelity ratio (i.e. the initial rate ofligating a substrate with an analogue located three nucleotides in fromthe 3′ end as well as a perfect match at the 3′ end divided by theinitial rate of ligating a substrate with an analogue located threenucleotides in from the 3′ end as well as a mismatch at the 3′ end) asfollows:${\frac{\left\lbrack k_{1} \right\rbrack_{{{SLP3}^{\prime}{QTT}},{match}}}{\left\lbrack k_{1} \right\rbrack_{{{SLP3}^{\prime}{QTT}},{mismatch}}} > \frac{\left\lbrack k_{1} \right\rbrack_{{{SLP3}^{\prime}{TTT}},{match}}}{\left\lbrack k_{1} \right\rbrack_{{{SLP3}^{\prime}{TTT}},{mismatch}}}} = {{Fidelity}\quad {ratio}}$

The above may be restated more generally to include other nucleotideanalogue or sugar phosphate backbone modifications as follows:${\frac{\left\lbrack k_{1} \right\rbrack_{{{Modified}\quad {oligo}},{match}}}{\left\lbrack k_{1} \right\rbrack_{{{Modified}\quad {oligo}},{mismatch}}} > \frac{\left\lbrack k_{1} \right\rbrack_{{{Unmodified}\quad {oligo}},{match}}}{\left\lbrack k_{1} \right\rbrack_{{{Unmodified}\quad {oligo}},{mismatch}}}} = {{Fidelity}\quad {ratio}}$

In the above equation, [k₁]_(Modified oligo, match) represents theinitial rate constant for ligating the first and second oligonucleotideprobes hybridized to a target nucleotide sequence wherein oneoligonucleotide probe contains a modification as well as having aperfect match at the ligation junction between the target nucleotidesequence and the oligonucleotide probe having its 3′ end at the ligationjunction. [k₁]_(Modified oligo, mismatch) represents the initial rateconstant for ligating the first and second oligonucleotide probeshybridized to a target nucleotide sequence wherein one oligonucleotideprobe contains a modification as well as having a mismatch at theligation junction between the target nucleotide sequence and theoligonucleotide probe having its 3′ end at the ligation junction.[k₁]_(Unmodified oligo, match) represents the initial rate constant forligating the first and second oligonucleotide probes hybridized to atarget nucleotide sequence with a perfect match at the ligation junctionbetween the target nucleotide sequence and the oligonucleotide probehaving its 3′ end at the ligation junction.[k₁]_(Unmodified oligo, mismatch) represents the initial rate constantfor ligating the first and second oligonucleotide probes hybridized to atarget with a mismatch at the ligation junction between the targetnucleotide sequence and the oligonucleotide probe having its 3′ end atthe ligation junction. For the modified oligonucleotide probe,[k₁]_(Modified oligo, match) divided by [k₁]_(Modified oligo, mismatch)(=fidelity ratio) is greater than the fidelity ratio for thecorresponding unmodified oligonucleotide probe. All modifiedoligonucleotide probes which satisfy the equation above will giveincreased discrimination of cancer-associated mutations in the presenceof an excess of normal DNA.

Another explanation for the above equation is the oligonucleotide probewhich has its 3′ end at the ligation junction has one or moremodification which differentially alters the rate of ligation when thefirst and second oligonucleotide probes hybridize to a minority targetnucleotide sequence with a perfect match at the ligation junctionbetween the minority target nucleotide sequence and the oligonucleotideprobe having its 3′ end at the ligation junction, compared to the rateof ligation when the first and second oligonucleotide probes hybridizeto the majority target nucleotide sequence with a mismatch at theligation junction between the majority target nucleotide sequence andthe oligonucleotide probe having its 3′ end at the ligation junction.

This can also be stated more generally in terms of a signal-to-noiseratio defined as follows:$\frac{\left\lbrack {{LDR}\quad {product}} \right\rbrack_{{minority}\quad {target}} + \left\lbrack {{LDR}\quad {product}} \right\rbrack_{{majority}\quad {target}}}{\left\lbrack {{LDR}\quad {product}} \right\rbrack_{{majority}\quad {target}}} = {{Signal}\text{-}{to}\text{-}{noise}\quad {ratio}}$

In the above equation, [LDR product]_(minority target) represents theamount of ligation product sequences produced when the first and secondoligonucleotide probes hybridize to a minority target nucleotidesequence with a perfect match at the ligation junction between theminority target nucleotide sequence and the oligonucleotide probe havingits 3′ end at the ligation junction. [LDR product]_(majority target)represents the amount of ligation product sequences produced when thesame first and second oligonucleotide probes hybridize to the majoritytarget nucleotide sequence with a mismatch at the ligation junctionbetween the majority target nucleotide sequence and the oligonucleotideprobe having its 3′ end at the ligation junction. The ligase, usingeither modified or unmodified oligonucleotide probes, has asignal-to-noise ratio, for the amount of ligation product sequencesproduced from both the minority and majority target nucleotide sequencesdivided by the amount of ligation product sequences produced from thesame amount of majority target nucleotide sequence alone.

When using thermostable ligase with both modified and unmodifiedoligonucleotide probes, there are signal-to-noise ratios associated witheach probe for detection of minority mutations, and the third aspect ofthe present invention can be expressed as the signal-to-noise ratioobtained using modified oligonucleotide probes is greater than thesignal-to-noise ratio obtained using unmodified oligonucleotide probes.$\frac{\frac{\left\lbrack {{LDR}\quad {product}} \right\rbrack_{{minority}\quad {target}} + \left\lbrack {{LDR}\quad {product}} \right\rbrack_{{majority}\quad {target}}}{\left\lbrack {{LDR}\quad {product}} \right\rbrack_{{majority}\quad {target}}}{Modified}\quad {Oligo}}{\frac{\left\lbrack {{LDR}\quad {product}} \right\rbrack_{{minority}\quad {target}} + \left\lbrack {{LDR}\quad {product}} \right\rbrack_{{majority}\quad {target}}}{\left\lbrack {{LDR}\quad {product}} \right\rbrack_{{majority}\quad {target}}}{Unmodified}\quad {Oligo}} > 1$

The above equation may be restated more simply as:$\frac{{Signal}\text{-}{to}\text{-}{noise}\quad {ratio}\quad {for}\quad {modified}\quad {oligonucleotide}}{{Signal}\text{-}{to}\text{-}{noise}\quad {ratio}\quad {for}\quad {unmodified}\quad {oligonucleotide}} > 1$

Ligation using the modified oligonucleotide probe has a signal-to-noiseratio, of the amount of LDR product produced from both the minority andmajority target nucleotide sequences, to the amount of LDR productproduced from the same amount of majority target nucleotide sequencealone, which is greater than the signal-to-noise ratio for ligationusing an oligonucleotide probe lacking the modification. All modifiedoligonucleotide probes which satisfy the equation above will giveincreased discrimination of cancer-associated mutations in the presenceof an excess of normal DNA.

Suitable modifications include the use of nucleotide analogues, such as1-(2′-Deoxy-β-D-ribofuranosyl)imidazole4-carboxamide,1-(2′-Deoxy-β-D-ribofuranosyl)-3-nitropyrrole,4-(2′-Deoxy-β-D-ribofuranosyl)imidazole-2-carboxamide,2′-Deoxy-5-fluorouridine, 2′-Deoxyinosine,6-(2′-Deoxy-β-D-ribofuranosyl)-6H,8H-3,4-dihydropyrimido[4,5-c][1,2]oxazine-7-one,2-Amino-7-(2′-deoxy-β-D-ribofuranosyl)-6-methoxyaminopurine,1-(2′-Deoxy-β-D-ribofuranosyl)-5-nitroindole,1-(2′-Deoxy-β-D-ribofuranosyl)pyrazole4-carboxamide,1-(2′-Deoxy-β-D-ribofuranosyl)-1,2,4-triazole-3-carboxamide,3-Amino-1-(2′-deoxy-β-D-ribofuranosyl)-1,2,4-triazole,5-(2′-Deoxy-β-D-ribofuranosyl)-2-pyrimidinone,5-(2′-Deoxy-β-D-ribofuranosyl)-2-thiopyrimidine,5-Amino-β-(2′-deoxy-β-D-ribofuranosyl)imidazole-4-carboxamide,1-(2′-Deoxy-β-D-ribofuranosyl)-3-nitropyrazole,1-(2′-Deoxy-β-D-ribofuranosyl)-4-iodopyrazole,1-(2′-Deoxy-β-D-ribofuranosyl)-4-propynylpyrazole,1-(2′-Deoxy-β-D-ribofuranosyl)pyrrole-3-carboxamide,1-(2′-Deoxy-β-D-ribofuranosyl)pyrazone-4-carboxamide,1-(2′-Deoxy-β-D-ribofuranosyl)-4-nitroimidazole, or1-(2′-Deoxy-β-D-ribofuranosyl)-4-nitropyrazole. Alternatively, themodified oligonucleotide probe contains thiophosphate, dithiophosphate,2′-methoxy, or 3′-amino-2′,3′-dideoxy-modifications to the sugarphosphate backbone of the oligonucleotide probe. This modification iseither at the position which undergoes ligation, the adjacent position,or the third position for that undergoing ligation.

EXAMPLES Example 1

Construction of Thermus thermophilus DNA Ligase Mutants at Amino AcidResidue 294 Using Site-Specific Mutagenesis

Thermus thermophilus (“Tth”) DNA ligase mutants were created using atwo-step PCR method (Horton et al., “Engineering Hybrid Genes Withoutthe Use of Restriction Enzymes: Gene Splicing By Overlap Extension,”Gene, 77:61-68 (1989), which is hereby incorporated by reference).Plasmid pDZ15 (linearized by HindIII) was used as a template in thefirst round of PCR reactions. In the upper panel of FIG. 10, plasmidpDZ15 which contains the cloned Tth DNA ligase gene (SEQ. ID. NO: 97)(Barany, F., et al., “Cloning, Overexpression, and Nucleotide Sequenceof a Thermostable DNA Ligase Gene,” Gene, 109:1-11 (1991), which ishereby incorporated by reference), is shown schematically as if openedat a PstI site with genes drawn approximately to scale. In pDZ15, theTth ligase gene and direction is represented by the strongly hatchedarrow; the vector Ap^(R) (bla) gene represented by the stippled (gray)arrow; the truncated end of a nonfunctional Taq I endonuclease generepresented by the lightly hatched arrow, and the pBR origin ofreplication represented by the open bar. The phoA, and T7 promoters areindicated by right angle arrows and point in the direction oftranscription. Restriction sites are: Av, AvrII; Bm, BamHI, Bg, BglII,(Bg/Bm recombined site is not cleavable by either BamHI or BglIIH;) Hd,HindIII; RI, EcoRI; Ps, PstI, Pv, PvuII. Polylinker regions from pTZ18Rare indicated by the triangular “rake”, with only the outsiderestriction sites listed. Escherichia coli host strains used in theconstructions described below were obtained from the following sources:N3098 (ligts7; (Wilson, G. G., et al., “Molecular Cloning of the DNALigase Gene From the Bacteriophage T4.I. Characterization of theRecombinants,” J. Mol. Biol., 132:471-491 (1979), which is herebyincorporated by reference), from N. Murray; JH132 (mrr⁻, Tn10; (Heitman,J., et al., “Site-Specific Methylases Induce the SOS DNA Repair Responsein Escherichia coli,” J. Bacteriol, 169:3243-3250 (1987), which ishereby incorporated by reference), from J. Heitman; MM294 (endA⁻, hsdR⁻,hsdM⁺, thi-1, supE44; (Miller, J. H., “Experiments in MolecularGenetics,” Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., pp.201-205 (1972), which is hereby incorporated by reference), from ourcollection. Strains AK53 (mrrB⁻, MM294) and AK76 (mrrB⁻, N3098) wereconstructed by transducing the mrrB⁻ phenotype from JH132 as described(Miller, J. H., “Experiments in Molecular Genetics,” Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y., pp. 201-205 (1972), which ishereby incorporated by reference). Presence of the mrrB⁻ phenotype wasconfirmed by tolerance of these strains to the Taq MTase-encoding genepresent on plasmid pFBT71 (Barany, F., “A Genetic System for Isolationand Characterization of TaqI Restriction Endonuclease Mutants,” Gene,56:13-27 (1987) and Barany, F., et al., “Cloning and Sequencing of theTthHB8I DNA Restriction and Modification Enzymes, and Comparison Withthe Isoschizomeric TaqI Enzymes,” Gene, 112:3-12 (1992), which is herebyincorporated by reference).

In the lower panel of FIG. 10, the Tth DNA ligase gene and its directionof transcription is represented by a hatched arrow. The cleavage sitesof some restriction endonucleases in the Tth DNA ligase gene areindicated by short solid bars. Approximate positions of oligonucleotideprimers used for PCR reactions are depicted by arrows and primer namesabove the Tth ligase gene. The site of mutation, amino acid residue K294is also indicated.

Site-directed mutagenesis at residue K294 was carried out as follows:(1) With HindIII-linearized pDZ15 as the template, two independent PCRreactions were performed. In one reaction tube, 400 ng of primers JL505and JL507R were added to 200 ng of HindIII digested pDZ15 containing 50μmoles of DATP, dCTP, dGTP, and dTTP each, and 2.5 units Amplitaq™ in100 μl PCR buffer and cycled as described (Saiki, R. K., et al.,“Primer-Directed Enzymatic Amplification of DNA With a Thermostable DNAPolymerase,” Science, 239:487-491 (1988), which is hereby incorporatedby reference); Perkin-Elmer Cetus Instruments, Emoryville Calif.) Asecond reaction tube contained 400 ng of primers JL506 and JL508R, 200ng of HindIII digested pDZ15, and 2.5 units Amplitaq™ in the samereaction buffer, and incubated as above. (2) A third PCR reaction wasthen carried out in 100 μl PCR buffer containing 1 μl of the productsfrom the two initial reactions, 400 ng primers JL505 and JL508 and 2.5units Amplitaq™ and incubated as above. After removal of Amplitaq™ thelarger product PCR fragment was digested with AvrII and BamHI, andelectrophoresed in low melting agarose. The 436 bp AvrII-BamHI fragmentwas excised from the gel, and purified as described previously (Barany,F., “Overproduction, Purification, and Crystallization of TaqIRestriction Endonuclease,” Gene, 63:167-177 (1988), which is herebyincorporated by reference). In addition, plasmid pDZ15 was also digestedwith AvrII and BamHI, and electrophoresed in low melting agarose. Thebigger fragment, which equals pDZ15 minus the 436 bp AvrII-BamHIfragment, was excised and purified. This big fragment was incubated withthe 436 bp AvrII-BamHI fragment purified from the product of the thirdPCR reaction in the presence of T4 DNA ligase for 16 hours at 14° C. Theligation mixture was transformed into E. coli strain AK76 (ts lig) asdescribed previously (Hanahan, D., “Studies on Transformation of E. coliWith Plasmids,” J. Mol. Biol., 166:557-580 (1983), which is herebyincorporated by reference). Plasmid carrying cells were replica platedonto Fortified Broth plates supplemented with either high concentrationof phosphates (10 mM K₂HPO₄, pH 7.6) or low concentration of phosphates(0.2 mM K₂HPO₄, pH 7.6) grown at 32° C. and 42° C. to test thecomplementation ability of mutant Tth DNA ligase to the ts lig host.Independent clones were picked from FB-high phos plates, and grown inliquid Fortified Broth supplemented with 10 mM K₂HPO₄, pH 7.6. Plasmidminipreps were made using the Magic Miniprep kit from Promega, and theAvrII-BamHI region was sequenced to confirm site-specific mutations atK294 using the Prism Dye DeoxyTerminator Cycle sequencing kit and DNAsequencer 373A from Applied Biosystems Division of the Perkin-ElmerCorporation.

Example 2

Construction of Tth DNA Ligase Mutants K294R and K294P

The mutant ligases of FIG. 11 were prepared using the oligonucleotidesof FIGS. 12A-B as described infra. Four oligonucleotide primers for PCRreactions were designed for creating multiple site-specific mutations atK294 site. Their sequences are as follows: Primer a (JL505) (SEQ. ID.No. 78): 5′CAG AAC CTC CTC ACC ATC 3′; Primer b (JL507R) (SEQ. ID. No.79): 5′CTC GTC CAG (G,C) (T, G, C, A) G CAC CAC CAC CCC GTC 3′; Primer c(JL506) (SEQ. ID. No. 88): 5′ TGG TGG TGC (A, C, G, T) (C, G) C TGG ACGAGC TTG CCC T 3′; and Primer d (JL508R) (SEQ. ID. No. 96): 5′ CTC TATGTA GCT CTC GTT GTG 3′. Primers b and c are overlapping primerscontaining degenerate codons at mutation site. These primers weresynthesized using reagents and a 394 automated DNA synthesizer fromApplied Biosystems Division of Perkin-Elmer Corporation, Foster City,Calif. After synthesis, primers were deprotected in 30% ammoniumhydroxide at 55° C. for 12 hours, dried in speedvac, resuspended in 100μl of ddH₂O (i.e. double distilled water), and purified by ethanolprecipitation. The pellet was resuspended in 200 μl of ddH₂O, and theirconcentrations were determined by spectrophotometry at OD₂₆₀. Primerswere then aliquoted and stored in a −20° C. freezer before use.

Site-specific mutagenesis in Tth DNA ligase gene (Tth lig) was carriedout using a two-stage PCR-based overlap extension strategy as describedpreviously (Ho et al., Gene 77:51-59 (1989), which is herebyincorporated by reference). Plasmid pDZ15, the expression plasmid(Barany, et al., Gene 109:1-11 (1991), and Horton, et al., “EngineeringHybrid Genes Without the Use of Restriction Enzymes: Gene Splicing byOverlap Extension,” Gene 77:61-68 (1989), which are hereby incorporatedby reference) of Tth DNA ligase was linearlized with a restrictionendonuclease HindIII, and used as the template for the first round ofPCR reactions. Two separate first round PCR reactions were carried outusing primers a (JL505) and b (JL507R) or primers c (JL506) and d(JL508R), respectively. One microliter of the product from each firstround PCR reaction was used as the template for the second round PCRreaction with primers a (JL505) and d (JL508R). The resulting productwas digested with restriction endonucleases AvrII and BamHI, andseparated on SeePlaque low-melting agarose gel. The DNA fragmentsassumed to contain the mutation site were cut from the low-meltingagarose gel, purified by phenol extraction before ligated to the biggerfragment created from pDZ15 by digestion with AvrII and BamHI. Theligation reaction was carried out at 14° C. for 16 hours. The resultingligation mixture was used to transform AK76, a bacterial strain (lig ts7strain, Barany, et al., Gene 109:1-11 (1991), which is herebyincorporated by reference) which contains a temperature-lethal mutationin ligase gene on bacterial chromosome. Positive transformants wereselected by growing transformants on TY plates containing 50 μg/mlAmpicillin. Plasmid DNA minipreps were made from the transformants usingthe Magic Minipreps columns from Promega, and used for sequencing.Regions which was amplified in PCR reactions were sequenced to confirmthe mutations using the Prism Dye DeoxyTerminator Cycle sequencing kitand DNA sequencer 373A from Applied Biosystems Division of Perkin-ElmerCorporation, Foster City, Calif.

Example 3

Expression of Mutant Tth DNA Ligases in E. coli

Plasmids containing mutant Tth DNA ligase gene under control of a phoApromoter were introduced into E. coli strain, AK53 via transformation.Mutant Tth DNA ligase proteins were overexpressed at 30° C. for 15 hoursin 6 ml MOPS medium (Neidhardt, et al., J. Bacteriol., 119:736-47(1974), which is hereby incorporated by reference) containing 0.2 mMK₂HPO₄ and 75 μg/ml ampicillin (F. Barany, et al., Gene 109:1-11 (1991),which is hereby incorporated by reference). Cells were harvested bycentrifugation, resuspended in 400 μl TE, (10 mM Tris, pH 8.5, 1 mMEDTA) sonicated for 3×10 seconds with a microprobe on a Sonifier 350cell disruptor from VWR, and centrifuged for 10 min. at 4° C. Thesupernatant was adjusted to 20 mM Tris-HCl, pH 8.5, 50 mM KCl, 10 mMMgCl₂, and 0.5 mM EDTA, 1 mM DTT, and 2 mM 2-mercaptoethanol. Afterincubation at 64° C. for 25 min, the cloudy suspension was clarified bycentrifugation at 4° C. for 15 min. Over 70% of the total protein in theresulting clear supernatant is Tth DNA ligase, as determined by stainingof a 7.5% polyacrylamide gel containing 0.1% SDS with CoomassieBrilliant Blue. Approximately 200 μg of Tth DNA ligase was isolated froma 6 ml culture. Every mutant Tth DNA ligase was overexpressed in AK53,and remained soluble after heat treatment at 65° C.

Example 4

Complementation Assay

Plasmids containing mutated Tth DNA ligase genes were introduced into atemperature sensitive ligase defective E. coli strain, AK76 (lig ts7strain) (F. Barany, et al., Gene 109:1-11 (1991), which is herebyincorporated by reference) via transformation. Individual transformantswere replica plated onto high phosphate plates (0.6% NaCl, 2.5%Bacto-Tryptone, 0.75% yeast extract, 0.1% dextrose, and 10 mM K₂HPO₄, pH7.6, and 50 μg/ml ampicillin), and low phosphate plates (0.2 mM K₂HPO₄)(Neidhardt, et al., J. Bacteriol., 119:736-47 (1974), which is herebyincorporated by reference); and incubated overnight at permissive (32°C.) and non-permissive (42° C.) temperatures, respectively. The Tth DNAligase gene is induced only at low phosphate concentration. An activeenzyme encoded by the plasmid enables the temperature sensitive host togrow at 42° C. on low phosphate plates.

Example 5

Adenylation Assays

Adenylation was assayed by incubating approximately 8 μg (100 fmoles)wild-type and mutant Tth DNA ligase, prepared as described above, in 100μl of reaction buffer containing 20 mM Tris-HCl, pH 7.6, 50 mM KCl, 10mM MgCl₂, 0.5 mM EDTA, 1 mM NAD⁺, 1 mM DTT, 2 mM 2-mercaptoethanol at65° C. for 25 min. Under these conditions, virtually all the wild-typeligase was found in the adenylated form. The reaction was stopped byadding an equal volume of 2×sample buffer containing 120 mM Tris-HCl, pH7.6, 2% SDS, 20% glycerol, 0.02% bromophenol blue, and 300 mM2-mercaptoethanol. Samples were boiled for 5 min, and were analyzed byloading 50 μl on to a 7.5% polyacrylamide-0.1%-SDS gel. The adenylatedenzyme can be distinguished by its higher apparent molecular weight (81Kd), compared to the deadenylated form (78 Kd).

To rule out the possibility that some mutants may not change mobilityafter adenylation, experiments were also performed with radioactive[³²P] NAD⁺. In this case, the reaction was carried out in 25 μl reactionmixture under the same conditions described above except that 3.3 μCi[³²P] NAD⁺ (800 Ci/mmol, NEN-Du Pont Company, Chadds Ford, Pa.) wasused. After 15 min incubation at 65° C., 1.5 pmoles of non-radioactiveNAD⁺ was added to the reaction mixture, and incubated for 5 more min todrive the adenylation reaction to near completion. Reactions werestopped by adding 25 μl of 2×sample buffer. The resulting mixture wasboiled at 100° C. for 5 min prior to analysis on a 7.5%polyacrylamide-0.1% SDS gel. The gel was autoradiographed at roomtemperature for 3 hours against a Kodak XAR-5 film. In order to verifyprotein samples in each lane, the gel was then stained with CoomassieBrilliant Blue.

Example 6

Deadenylation Assays

To assay for the deadenylation activity (transfer of the adenyl groupfrom enzyme to DNA substrate), the same conditions were used as for theadenylation experiment, except that 1 mM NAD⁺ was replaced by 5 μg ofnicked salmon sperm DNA; prepared by incubating salmon sperm DNA (Sigma,St. Louis) with pancreatic DNase I (Barany, et al., Gene 109:1-11(1991), which is hereby incorporated by reference). The deadenylatedenzyme was recognized as a fast migrating band (78 Kd) when separated byelectrophoresis on a 7.5% polyacrylamide-0.1%-SDS gel.

Example 7

5′-Labeling of Oligonucleotide Probe with [γ-³²P] ATP

Oligonucleotide probe JL514 was 5′ labeled in a 10 μl reactioncontaining 50 mM Tris-HCl, pH 7.6, 10 mM MgCl₂, 1 mM EDTA, 10 mM DTT, 45pmole of [g-³²P] ATP (6000 Ci/mmol, NEN-Du Pont Company), 15 pmole ofgel-purified oligonucleotides, and 10 units of T4 polynucleotide kinase(New England Biolabs, Beverly, Mass.) after incubation at 37° C. for 45min. 1 μl of 10 mM ATP was added to the reaction mixture, and theincubation was continued for two more minutes. The reaction was quenchedby adding 17 μl of 60 mM EDTA. The kinase was heat-inactivated byincubation at 64° C. for 20 min. Phosphorylated oligonucleotides wereseparated on a Sephadex G-25 column equilibrated in TE buffer. Fractionscontaining phosphorylated oligonucleotides were combined, and stored at−20° C. in aliquots before use. The specific radioactivity ofphosphorylated oligonucleotide JL514 was 9×10⁶ cpm/pmol.

Example 8

Assay for Nick-Closure Activity

The nicked DNA duplex substrate was formed by annealing two shortoligonucleotide probes (JL538 and JL514) to a longer complementaryoligonucleotide target (JL539). Their nucleotide sequences are: JL538(SEQ. ID. No. 75): 5′ AAC CAC AGG CTG CTG CGG ATG CCG GTC GGA G 3′;JL514 (SEQ. ID. No. 76): 5′ AGA GCC GCC ACC CTC AGA ACC GCC ACC CTC 3′;JL539 (SEQ. ID. No. 77): 5′ GAG GGT GGC GGT TCT-GAG GGT GGC GGC TCT CTCCGA CCG GCA TCC GCA GCA GCC TGT GGT T 3′. The reaction was carried outin 40 μl of buffer containing 20 mM Tris-HCl, pH 7.6, 10 mM MgCl₂, 100mM KCl, 10 mM DTT, 1 mM NAD⁺, and 60 fmol of nicked DNA duplexsubstrates. DNA probes and target were denatured by incubating thereaction mixture at 94° C. for 2 min, and re-annealed at 65° C. for 2min. Ligation reactions were initiated by addition of Tth DNA ligase andcarried out at 65° C. for 30 min. Reactions were terminated by adding 40μl of stop solution (83% formamide, 8.3 mM EDTA, and 0.17% bluedextran). Samples were denatured at 93° C. for 2 min, chilled rapidly onice prior to loading 20 μl on an 8 M urea-10% polyacrylamide gel. Afterelectrophoresis, the gel was exposed to a phosphorimager screen for 20min. Radioactively labeled ligation products were analyzed on aMolecular Dynamics Phosphorimager (Sunnyvale, Calif.) and quantifiedusing Image-Quant software.

The amino acid sequence of the Tth DNA ligase gene contains two shortsequences, K¹¹⁸VDG and DGVVVK²⁹⁴, which resemble the active sitesequence (KYDGQR) of human DNA ligase I. Since human DNA ligase Irequires ATP as a cofactor while the Tth DNA ligase uses NAD⁺ instead,it is possible that their active sites for enzyme-adenylate formationmay differ. Although, the sequence K¹¹⁸VDG resembles the active sitesequence (KY/LDGXR) of human DNA ligases I, III and IV more than thesequence DGVVVK²⁹⁴ does, both sequences were tested for adenylation.

Site-specific mutants were constructed at three amino acid sites, K118,D120, and K294 (FIG. 11). At least four different single amino acidsubstitutions were made at each site to explore a range of side chainchanges (FIG. 11). Mutant Tth DNA ligases were overexpressed in AK53cells, partially purified, and analyzed on a 7.5%polyacrylamide-0.1%-SDS gel (Barany, et al., “Cloning, Overexpression,and Nucleotide Sequence of a Thermostable DNA Ligase Gene,” Gene109:1-11 (1991), which is hereby incorporated by reference). E. colicells transformed with the plasmid vector lacking the Tth DNA ligasegene showed no protein in the molecular weight range of Tth DNA ligase(76-81 Kd), although some heat-resistant impurities from the hostbacteria are visible. Wild-type Tth DNA ligase and mutant ligases withamino acid substitution at K294 migrate as doublet bands duringelectrophoresis. The upper band, with an apparent molecular weight of 81Kd is the adenylated form while the lower one at 78 Kd is thedeadenylated form. Id. Nine out of ten mutant ligases of K118 and D120migrated as a single band. The exception was D120E, the majority ofwhich was expressed as the adenylated form while a very small amount ofthe deadenylated form was seen.

Partially purified mutant Tth DNA ligases were assayed for adenylationin the presence of NAD⁺, and deadenylation in the presence of nickedsalmon sperm DNA. Both wild-type enzyme and all K294 mutants testedbecame adenylated in the presence of NAD⁺ and deadenylated uponincubation with nicked salmon sperm DNA (See Table 1).

TABLE 1 Effects of amino acid substitutions at K118, D120, and K294 ofTth DNA ligase on enzyme activities^(a) Complementation Nick-closure ofts7 lig activity (%) Plasmid Mutant Adenylation Deadenylation (in vivo)(in vitro) pDZ15 Wildtype + + + 100 pJLBE3 K118R − NA − ND pJLBE12 K118H− NA − ND pJLBE5 K118L − NA − ND pJLBE9 K118P1 (CCC) − NA − ND pJLBE11K118P2 (CCG) − NA − ND pJLBg3 D120E + ± − 6.2 pJLBF9 D120N + ± − 8.5pJLBF7 D120Y ± − − 0.11 pJLBc4 D120G ± − − 0.07 pJLBd6 D120A ± − − 0.48pJLBf6 D120V − NA − ND pJLBH7 K294R + + + 100 pJLBH2 K294Q + + + 77pJLBH6 K294L1 (CTG) + + + 90 pJLBH10 K294L2 (CTC) + + + 87 pJLBH8K294P + + + 26 pJLBH9 K294P* + + − ND ^(a)Abbreviations: (+), similaractivity to wildtype enzyme; (−), no activity; (±), intermediateactivity; Deadenylation refers to transfer of the adenyl group fromenzyme to nicked DNA substrate; NA, “Deadenylation” could not bedetermined since these mutants do not adenylate; ND, not detectable;(*), Mutant encoded in plasmid pJLBH9 also contains the G339E secondsite mutation. For the nick-closure activity experiment, 100% activityrepresents the formation of 58 # fmol product in 30 min. at 65° C. using6 fmol of partially purified wild type Tth DNA ligase under conditionsdescribed herein. The amount of partially purified mutant enzymes usedin this experiment were: 60 fmol for mutants at K118 and D120 sites, 6fmol for mutants at K294 site, and 60 fmol for mutant K294P*(K294P/G339E). The relative nick-closure activity for mutant Tth DNAligases shown in this Table was normalized based on the amount of enzymeused.

All mutants at K118 site were unchanged by treatments with either NAD⁺or nicked salmon sperm DNA, indicating a possible defect in theadenylation or deadenylation reaction. Likewise, mutant D120V was alsodefective in adenylation or deadenylation (Table 1). Mutants D120A,D120G, D120Y, and D120N underwent adenylation (see below), although themobility shifts were difficult to distinguish for some of the mutants.Most of the mutant D120E protein remained in the adenylated form whenisolated from E. coli cells, indicating a possible defect during thedeadenylation step. When 5 μg of mutant D120E and wild-type enzyme wereincubated with 2.5 μg of nicked salmon sperm DNA, wild-type enzymebecame completely deadenylated, while most of D120E remained as theadenylated form (data not shown). However, mutant D120E wassubstantially deadenylated when the amount of nicked salmon sperm DNAwas raised above 25 μg. It is unlikely that this change is due to thereversal of the adenylation step since no β-nicotinamide mononucleotide(NMN) was added in the reaction mixture. Thus, mutant D120E either has areduced affinity to the DNA substrate, or has a reduced rate oftransferring the AMP moiety to the 5′ phosphate of the DNA substrate.

To rule out the possibility that some mutants may become adenylatedwithout altering mobility on SDS gels, adenylation was also carried outusing [³²P] NAD⁺. None of the K118 mutants were adenylated when assayedwith radioactive substrate, while the wild-type enzyme yielded a strongradioactive adenylated-enzyme band. This indicates that K118 isessential for enzyme-adenylate formation. All mutants at D120 siteexcept D120V had incoporated a comparable amount of radioactive AMPrelative to that by wild type enzyme. However, when the gel was stainedwith Coomassie Blue after autoradiography, mutant proteins D120Y andD120G showed only partial adenylation, indicating the higher sensitivityof the radioactive assay. Mutant D120A formed the enzyme-adenylatewithout changing its electrophoretic mobility, while mutants K118P1 andK118P2 changed mobility not as a result of adenylation, but due to aconformational change caused by the proline for lysine substitution.Thus, a conformational change (as evidenced by altered mobility on anSDS gel) is usually observed upon successful adenylation, but may alsobe achieved by some of the same mutations which abolish formation of theenzyme-adenylate complex.

The effects of amino acid substitution at K118, D120, and K294 on theoverall activity of Tth DNA ligase were tested by a complementationassay and an in vitro ligation assay (Table 1). In this in vitro assay,varying concentrations of wild-type and mutant Tth DNA ligases wereincubated with a nicked-DNA duplex substrate (composed of two probeshybridized to a synthetic target), and ligation product separated on adenaturing gel. Wild-type Tth DNA ligase complemented the E. coli ligts7 host while none of the mutants at K118 and D120 did. All K118mutants were also defective for in vitro ligation activity, butunexpectedly, several D120 mutants retained some in vitro activity(Table 1). Mutants D120E and D120N had 6.2% and 8.5% activityrespectively, while D120Y, D120G, and D120A all had less than 0.5%activity. No in vitro activity was detected for D120V. All K294 mutantswith the exception of one double mutant K294P/G339E, supported the E.coli lig ts7 host growth at 42° C., as well as retaining significant invitro enzymatic activity. The aberrant clone did not complement the E.coli lig ts7 host, but showed normal activity for adenylation anddeadenylation. Sequencing this clone revealed a second mutation of G339Ein addition to K294P. The possible involvement of G339 in the formationof phosphodiester bonds was studied further and is discussed below.

The results on single amino acid substitutions of K118, D120, and K294indicate that K118 is critical for enzyme-adenylate formation, D120facilitates deadenylation, and K118VDG is thus inferred to be the siteof Tth DNA ligase-adenylate formation. This supports the prediction fromsequence alignment that KXDG may also be the active site ofNAD⁺-dependent DNA ligases. Similar results using site-directedmutagenesis were reported for ATP-dependent human DNA ligase I (Kodama,K., et al., Nucleic Acids Res., 19:6093-99 (1991), which is herebyincorporated by reference) and vaccinia DNA ligase (Cong, P., et al., J.Biol. Chem., 268:7256-60 (1993) and Shuman, S., et al., Virology,211:73-83 (1995), which are hereby incorporated by reference).Substitution of the active site Lys (K568) by His or Arg in human DNAligase I, and of K231 by Asn or Arg in vaccinia DNA ligase caused a lossof the adenylation activity. Mutations at the conserved Asp (D570) toAsn, Glu, and Gln in human DNA ligase I reduced enzyme-adenylateformation and caused loss of in vivo complementation (Kodama, K., etal., Nucleic Acids Res., 19:6093-99 (1991), which is hereby incorporatedby reference). A KEDG motif was identified as the active site of T4 RNAligase, based on mass spectrometry of an adenylated peptide (Thogersen,H. C., et al., Eur. J. Biochem., 147:325-29 (1985), which is herebyincorporated by reference), and site-directed mutagenesis studies(Heaphy, S., et al., Biochemistry, 26:1688-96 (1987), which is herebyincorporated by reference). In this enzyme, substitution of theconserved Asp (D101) by Asn, Ser, or Glu was well tolerated for enzyme-adenylate formation, while deadenylation and phosphodiester bondformation steps were prevented completely by each mutation. It wassuggested that this Asp residue interacts with the substrate5′-phosphate terminus rather than the substrate 3′-OH terminus or theadenylate group. All of our D120 substitutions also caused loss ofcomplementation activity (assayed in vivo at 42° C.), yet D120E andD120N still retained some in vitro ligation activity (assayed at 65° C.,see Table 1). Therefore, while D120 clearly facilitates deadenylation inTth DNA ligase, it is not strictly essential for ligation. This findingcorroborates a similar conclusion by Shuman and Schwer based on studiesof capping enzymes and ATP dependent ligases (Shuman, S., et al., Molec.Microbiol., 17:405-10 (1995), which is hereby incorporated byreference).

A KTDG motif was deduced to be the active site of the mRNA cappingenzymes of the Vaccinia virus (Cong, P., et al., J. Biol. Chem.,268:7256-60 (1993), which is hereby incorporated by reference), S.cerevisiae (Fresco, L. D., et al., Proc. Natl. Acad. Sci. USA,91:6624-28 (1994) and Schwer, B., et al., Proc. Natl. Acad. Sci. USA,91:4328-32 (1994), which are hereby incorporated by reference), and S.pombe (Shuman, S., et al., Proc. Natl. Acad. Sci. USA, 91:12046-50(1994), which is hereby incorporated by reference), for enzyme-guanylateformation. In Yeast tRNA ligase, the amino acid sequence KANG wasidentified by sequencing the adenylated peptide (Xu, Q., et al.,Biochemistry, 29:6132-38 (1990), which is hereby incorporated byreference). A comparison of 5 capping enzymes and 14 ATP dependent DNAand RNA ligases suggests a superfamily of five evolutionarily conservedmotifs which plays a role in nucleotidyl binding and transfer to an RNAor DNA substrate (Shuman, S., et al., Molec. Microbiol., 17:405-10(1995); Shuman, S., et al., Proc. Natl. Acad. Sci. USA, 91:12046-50(1994); and Cong, P., et al., Molec. Cell. Biol., 15:6222-31 (1995),which are hereby incorporated by reference). These earlier studies, plusthe present work on an NAD³⁰ requiring ligase, allow us to considerKXD/NG as a general active site motif for creating a chargedenzyme-nucleotide complex, which provides the energy to form a covalentphosphodiester bond in nucleic acid substrates.

The observation that the double mutant (K294P/G339E) lost ligaseactivity suggests that G339 may be important for the third step of theligation reaction; i.e. formation of the phosphodiester bond. To confirmthat this effect is caused by one mutation at G339 site, and not by anadditive effect of two mutations, single amino acid substitutions weremade at G339 by site-directed mutagenesis. Site-specific mutations werealso made at R337, a conserved positively charged amino acid near G339,and at C412, C415, C428, and C433 (FIG. 11). There are only four Cysresidues in Tth DNA ligase, all conserved among the five NAD⁺-dependentbacterial ligases that are sequenced. These four Cys residues may form azinc-binding site and be involved in the interaction between bacterialDNA ligase and DNA substrates (Thorbjamardottir, S. H., et al., Gene,161:1-6 (1995), which is hereby incorporated by reference).

All Tth DNA ligase mutants constructed at these six sites were able toform the enzyme-adenylate complex in the presence of NAD⁺, and weredeadenylated in the presence of nicked salmon sperm DNA (Table 2). Theeffects on the overall ligase activity varied with each mutation, butwas generally consistent, when comparing in vivo and in vitro activities(Table 2).

TABLE 2 Effects of single amino acid substitutions at R337, G339, C412,C415, C428, C433 of Tth DNA ligase on enzyme activity^(a)Complementation Nick-closure of ts7 lig activity (%) Plasmid MutantAdenylation Deadenylation (in vivo) (in vitro) pDZ15 Wildtype + + + 100pJLBK8 R337K + + + 3.1 pJLBK4 R337Q + + − 0.70 pJLBK5 R337E + + − 0.04pJLBJ6 G339A + + − 0.71 pJLBJ5 G339D + + − 0.34 pJLBA6 G339E + + − 0.22pJLBB4 C412A + + + 41 pJLBB9 C412V + + − 0.16 pJLBB11 C412T + + − 0.11pJLBB12 C412M + + − 0.17 pJLBC18 C415A + + + 79 pJLBC3 C415V + + + 100pJLBC9 C415T + + + 3.0 pJLBC19 C415M + + + 0.47 pJLBD10 C428A + + + 57pJLBD6 C428T + + − 0.43 pJLBI11 C433A + + − 0.15 pJLBI6 C433V + + − 0.02pJLBI4 C433T + + − 0.07 pJLBI1 C433M + + − 0.06 ^(a)Abbreviations: (+),similar activity to wildtype enzyme; (−), no activity; ND, notdetectable. For the nick-closure activity experiment, 100% activityrepresents the formation of 58 fmol product in 30 min. at 65° C. using 6fmol of partially purified wild type Tth DNA ligase or the formation of16.2 fmol product in 30 min at 65° C. using 0.6 fmol. The amount of theother enzymes used was either 0.6 fmol, 6 fmol, or 60 fmol. The relativenick-closure activity for # mutant Tth DNA ligases shown in this Tablewas normalized based on the amount of enzyme used.

Mutants R337Q and R337E lost activity and were unable to complement E.coli lig ts7 host, while mutant R337K retained partial in vitro activityand complementation activity. Substitution of G339 by Ala, Asp, or Gluall rendered the enzyme inactive, both in vivo and in vitro. At C412,substitution by Ala had no effect in both in vivo and in vitroexperiments, while substitution by Val, Thr, and Met caused the loss ofthe overall enzyme activity. Similar results were also observed at C428.Three of four mutations at C415 site (C415A, C415V, and C415T), allretained complementation and enzymatic activity. It is not clear whyC415M retained complementation activity (assayed at 42° C.) with suchpoor enzymatic activity (assayed at 65° C.), although the mutation mayhave rendered the enzyme thermostable. In contrast, all mutations atC433 site (C433A, C433V, C433T, or C433M) caused loss of bothcomplementation and enzymatic activity.

While the adenylation active site in many DNA ligases has been welldefined, the possible active site for formation of the phosphodiesterbond remains poorly understood. Residues G339 and C433 may be involvedin this third step of the ligation reaction, because conservativemutations at both sites abolished the overall enzyme activity withoutaffecting the first two steps, adenylation and deadenylation. ResidueG339 may allow a local structure critical for enzymatic activity, orinteract with the DNA substrate via the peptide backbone, in a mannerwhich would be incompatible with an Ala, Glu, or Asp substitution.Glycine residues play essential roles in mRNA capping enzymes, althoughthese mutations interfered primarily with enzyme-guanylate formation(Shuman, S., et al., Proc. Natl. Acad. Sci. USA, 91:12046-50 (1994) andCong, P., et al., Molec. Cell. Biol., 15:6222-31 (1995), which arehereby incorporated by reference). Residues R337, C412, and C428 mayplay an indirect, but not essential, role in the third step of ligationreaction since only nonconservative mutations caused loss of activityfor this step. For all mutants at these five residues, there were nodramatic global conformational changes induced by these mutations asindicated by their ability to form enzyme-adenylate complex in thepresence of NAD⁺, and to deadenylate in the presence of nicked DNAsubstrate. The active site for the third step of ligation may well beseparated from that of the first two steps. These active sites functionindependent of one another and damage at one site may not affect theactivity of the other.

In summary, site-directed mutagenesis studies were carried out toidentify active sites for Tth DNA ligase. The adenylation active siteand facilitated deadenylation site of Tth DNA ligase was identified asthe Lys and Asp residues of motif K¹¹⁸VDG. These results are consistentwith other mutagenesis studies on the active sites of DNA ligases(Kodama, K., et al., Nucleic Acids Res., 19:6093-99 (1991); Cong, P., etal., J. Biol. Chem., 268:7256-60 (1993); and Shuman, S., et al.,Virology, 211:73-83 (1995), which are hereby incorporated by reference),T4 RNA ligase (Heaphy, S., et al., Biochemistry, 26:1688-96 (1987),which is hereby incorporated by reference) and MRNA capping enzymes(Cong, P., et al., J. Biol. Chem., 268:7256-60 (1993); Fresco, L. D., etal., Proc. Natl. Acad. Sci. USA, 91:6624-28 (1994); Schwer, B., et al.,Proc. Natl. Acad. Sci. USA, 91:4328-32 (1994); Shuman, S., et al., Proc.Natl. Acad. Sci. USA, 91:12046-50 (1994); and Cong, P., et al., Molec.Cell. Biol., 6222-31 (1995), which are hereby incorporated byreference). Together, they support the idea that KXD/NG is the activesite motif for the formation of enzyme-nucleotide complex. Mutations atresidues G339 and C433 did not inhibit adenylation and deadenylationsteps but abolished the overall activity, indicating that these twoamino acid residues may be involved in the third step of ligation, theformation of the phosphodiester bond.

Example 9

Synthesis of Oligonucleotide Probes

Oligonucleotide probes were synthesized using reagents and a model 394automated DNA synthesizer from Applied Biosystems Division ofPerkin-Elmer Corporation, (Foster City, Calif.). Fluorescent label wasattached to the 5′ end of oligonucleotides using 6-FAM (6-carboxyfluorescein) amidites, or attached to a 3′-amino group (C3-CPG columnfrom Glen Research (Sterling, Va.)) using NHS-FAM (N-hydroxysuccinimideester of FAM) from Applied Biosystems Division of Perkin-ElmerCorporation. A universal nucleotide analogue,1-(2′-deoxy-b-D-ribofuranosyl)-3-nitropyrrole, herein designated as Q,was synthesized, transformed to the phosphoramidite, andoligonucleotides synthesized as described (Bergstrom, D. E., et al.,JACS, 117:1201-1209 (1995), which is hereby incorporated by reference).All oligonucleotides used in this study were purified by polyacrylamidegel electrophoresis with recovery of DNA from gel slices using C-18Sep-Pak Cartridges from Waters Division of Millipore (Bedford, Mass.).

Example 10

5′-Phosphorylation of Oligonucleotide Probes

One mmole of gel-purified oligonucleotide was phosphorylated in a 25 μlreaction containing 50 mM Tris-HCl, pH 7.6, 10 mM MgCl₂, 1 mM EDTA, 10mM DTT, 1 mM ATP, and 10 units of T4 polynucleotide kinase (New EnglandBiolabs, Beverly, Mass.) at 37° C. for 45 minutes. The reaction wasquenched by adding 0.5 μl of 0.5 M EDTA, and the kinase washeat-inactivated by incubation at 64° C. for 20 minutes. Thephosphorylated oligonucleotides were stored at −20° C. in 5 μl aliquotsbefore use.

Example 11

Purification of Wild-Type and Mutant Tth DNA Ligase

Wild-type Tth DNA ligase was purified from an E. coli strain containingthe Tth ligase gene underphoA promoter control as described (Barany, F.,et al., Gene, 109:1-11 (1991)), which is hereby incorporated byreference) with some modifications. Briefly, cells were grown overnightat 30° C. in low phosphate (inducing) medium, harvested, resuspended inlysis buffer (20 mM Tris-HCl pH 8.5, 1 mM EDTA, 10 mM 2-mercaptoethanol,0.15 mM PMSF) and sonicated (Barany, F., et al., Gene, 109:1-11 (1991),which is hereby incorporated by reference). After removal of cellulardebris, the supernatant was adjusted to 20 mM Tris-HCl pH 8.5, 50 mMKCl, 10 mM MgCl₂, 0.5 mM EDTA, 1 mM DTT, and 2 mM 2-mercaptoethanol,incubated at 65° C. for 30 min., and cleared by centrifugation at 4° C.The supernatant was diluted with an equal volume of 10 mM Tris-HCl, pH7.6, 0.5 mM EDTA, filtered, and loaded onto a 10 ml Red-Sepharose column(Pharmacia) equilibrated with 20 mM Tris-HCl, pH 8.5, 50 mM KCl, 1 mMEDTA, 20% glycerol. The protein was eluted with a 30 ml linear saltgradient of 50 mM to 1 M KCl (Takahaski, M., et al., Agric. Biol. Chem.,50:1333-1334 (1986), which is hereby incorporated by reference), usingan FPLC apparatus from Pharmacia. Tth DNA ligase eluted between 0.4-0.8M KCl, and fractions containing pure Tth DNA ligase (seen as a doubletof adenylated and deadenylated forms on Coomassie-stained 7.5%polyacrylamide-0.1% SDS polyacrylamide gels), were pooled. The enzymewas precipitated with an equal volume of saturated ammonium sulfate, thepellet dissolved in 1.5 ml dH₂O, and dialyzed at 4° C. against 500 mlstorage buffer containing 10 mM Tris-HCl, pH 8.5, 1 mM EDTA, 1 mM DTT,0.2 mg/ml BSA, and 50% glycerol. Protein concentration was determined bythe Bradford method (Bradford, M. M., Anal. Biochem., 72:248-254 (1976),which is hereby incorporated by reference). About 4 mg of Tth DNA ligasewas obtained from 450 ml culture. Mutant Tth DNA ligase was partiallypurified as described previously.

Example 12

Fidelity Assays of Nick Closure by Tth DNA Ligase

Each reaction was performed in 40 μl of buffer containing 20 mMTris-HCl, pH 7.6; 10 mM MgCl₂; 100 mM KCl; 10 mM DTT; 1 mM NAD⁺; and12.5 nM (500 fmoles) of nicked DNA duplex substrates. DNA probes weredenatured (94° C. for 2 min), re-annealed (65° C. for 2 min), andligations initiated by the addition of 0.125 nM (5 fmoles) Tth DNAligase and carried out at 65° C. Five μl aliquots were removed at 0 hr,2 hr, 4 hr, 6 hr, 8 hr, and 23 hr, and mixed with 18 μl of a stopsolution (83% formamide, 8.3 mM EDTA, and 0.17% Blue Dextran). To 5 μlof this mixture, 0.5 μl of ROX-1000, a fluorescently labeled in-lanesize standard (Applied Biosystems Division of Perkin-Elmer) was added.Samples were denatured at 93° C. for 2 min, rapidly chilled on ice priorto loading on an 8 M urea-10% polyacrylamide gel, and electrophoresed at1400 V (constant voltage) on a model 373A automated DNA Sequencer(Applied Biosystems Division of Perkin-Elmer Corporation).Electrophoresis conditions were modified as suggested by themanufacturer. The gel was polymerized in 1.2×TBE (54 mM Tris-Borate and1.2 mM EDTA, pH 8.0) and was pre-run before loading samples in a runningbuffer of 0.6×TBE (27 mM Tris-Borate and 0.6 mM EDTA, pH 8.0) for 30 minwith an electrode polarity opposite to the normal run with samples.After pre-run and sample-loading, the gel was run in 0.6×TBE in thenormal top to bottom direction for 2.5 hrs. Fluorescently labeledligation products were analyzed and quantified using Genescan 672version 1.2 software (Applied Biosystems Division of Perkin-ElmerCorporation), and the results were plotted using DeltaGraph Pro3software (DeltaPoint, Inc. Monterey, Calif.).

Example 13

Measurement of Initial Rates of Perfect Match and Mismatch Ligations byTth DNA Ligase

Conditions for these experiments were the same as that for the fidelityassay except that different amounts of Tth DNA ligase and differentprobes were used, as indicated in Brief Description of the Drawings.Reactions were carried out in 160 μl of reaction buffer containing 12.5nM (2 pmoles) of nicked DNA duplex substrates at 65° C. DNA probes andtarget were denatured by incubating the reaction mixture at 94° C. for 2min, and re-annealed at 65° C. for 2 min. Ligations were initiated bythe addition of the Tth DNA ligase. Aliquots (10 μl) were removed at 0,2, 4, 6, 8, and 10 hr for reactions containing mismatched substrates;and at 0, 10, 20, 30, 40, 50, 60, and 70 sec for reactions containingmatched substrates. For assays using matched substrates, 2.5 μl sampleswere mixed with 2.5 μl loading buffer, 0.5 μl of ROX-1000 before gelelectrophoresis. Since the linear detection range of fluorescent sampleson the 373A DNA Sequencer is from 0.1 fmol to 10 fmol, products frommismatch ligation with a yield less than 1% were concentrated by ethanolprecipitation for accurate quantification. From 10 μl aliquot, 9.5 μlwas brought up to 200 μl with TE buffer (10 mM Tris-HCl, pH 8.0, 1 mMEDTA) and ethanol precipitated with 4 μg of Yeast tRNA as carriers. Thepellet was resuspended in 5 μl loading buffer and 0.5 μl ROX-1000 beforegel electrophoresis. The amount of unreacted fluorescent probe wasdetermined by diluting 0.5 μl of the 10 μl aliquot with 4.5 μl loadingbuffer plus 0.5 μl ROX-1000. Samples were separated on denaturingpolyacrylamide gels, and results analyzed as described above. Theinitial rates were calculated as the slope of the straight line in thegraph with the x-axis as the time, and the y-axis as the yield ofproducts.

Example 14

Preparation of Oligonucleotide Probes Containing Base Analogues andMismatch in Third Position on the 3′ Side of the Nick

Oligonucleotide probes were designed to test the possibility ofimproving the fidelity of LDR reactions using the Tth DNA ligase byintroducing a base analogue or a mismatch at the third position on the3′-side of the nick. These ten descriminating probes were made in 5pairs. The two probes in each pair differ by one base at the 3′ end (“C”or “T”). The probe with the “C” at the 3′ end has two more bases at its5′ end than the “T” probe, allowing one to distinguish the ligationproducts on a sequencing gel. The base analogue “Q” and other bases atthe third position from the 3′ end are underlined. A nicked DNA duplexsubstrate is formed by annealing one of left side probes, (for example,SLP3′TTT), and the common fluorescently labeled probe Com 610-3F to atemplate probe (for example, GLg.m3A). This substrate contains a T-Gmismatch on the 3′ side of the nick. Accurate quantification of mismatchand perfect match ligation products can be achieved by scanning thefluorescently labeled products using Genescan program. A ratio ofinitial rates of perfect match ligation over mismatch ligation indicatesthe fidelity of the LDR reaction. An extra mismatch or a base analoguewas introduced in the third position on the 3′ side of the nick in orderto improve the fidelity. In FIG. 17A, probes SLP3′Q₂TC, SLP3′Q₂TT,SLP3′TTC, and SLP3′TTT were used on targets GLg.m3A to test thecontribution of Q₂:A to increased fidelity compared with a T:A match atthe 3rd position from the 3′-side. The SLP3′TTC and SLP3′TTT probes wereused on mixtures of targets GLg.m3A and ALg.m3A to test for detecting arare target (cancer mutation) in the excess of a common target (normalDNA). In FIG. 13B, probes SLP3′ATC and SLP3′ATT were used on targets GLgto test the contribution of A:C to increased fidelity compared with aG:C match at the 3rd position from the 3′-side. In FIG. 17C, probesSLP3′Q₂TC, SLP3′Q₂TT, SLP3′Q₁₈TC, SLP3′Q₁₈TT, SLP3′GTC, and SLP3′GTTwere used on target GLg.m3T to test the contribution of Q₂:T, Q₁₈:T orG:T to increased fidelity compared with a T:A match at the 3rd positionfrom the 3′-side.

Example 15

Fluorescent Assay

As shown in FIGS. 13A-C, one of the four long oligonucleotides GLg, ALg,TLg or CLg (shown in the FIG. 13C) was used as the template strand,which vary at the underlined base. FIGS. 13A-B represent the formationof nicked DNA duplex using one of the template strands, ALg, as anexample. Shown in FIG. 13A, 4 different nicked DNA substrates are formedby annealing the common fluorescently labeled oligo, com5F, and one ofthe discriminating oligos (RP5′A, RP5′C, RP5′G, RP5′T) to the templatestrand, ALg. In FIG. 13B, 4 different nicked DNA substrates are formedby annealing the fluorescently labeled oligo, com3F, and one of thediscriminating oligos (LP3′A, LP3′C, LP3′G, LP3′T) to the templatestrand, ALg. A matrix of nicked DNA duplexes is thus formed with allpossible combinations of match and mismatch base pairing on the 3′ andthe 5′ side of the nick, when ALg is replaced by one of the followingtemplate strands, GLg, TLg, and CLg. Products formed by ligation to thecommon fluorescently labeled probes can be discriminated by size ondenaturing polyacrylamide gel due to the incorporation of differentlength of “A” tails.

Sequences of these probes (shown in FIG. 14) were derived from that ofhuman eukaryotic protein synthesis initiation factor eIF-4E (Rychlik,W., et al., “Amino Acid Sequence of the mRNA Cap-Binding Protein FromHuman Tissue,” Proc. Natl. Acad. Sci. USA, 84:945-949 (1987), which ishereby incorporated by reference). A random DNA sequence from aeukaryotic source was chosen to avoid any false signal arising frompossible bacterial DNA contamination in Tth DNA ligase preparation. Themelting temperature of probes were predicted using the nearest neighborthermodynamic method (Breslauer, K. J., et al., “Predicting DNA DuplexStability From the Base Sequence,” Proc. Natl. Acad. Sci. USA,83:3746-3750 (1986), which is hereby incorporated by reference). OLIGO4.0 program from National Biosciences Inc., Plymouth, Minn. was used torule out possible hairpin structure, repetitive sequences, and falsepriming. The templates and detecting oligonucleotides for this assayhave been designed such that their melting temperature is sufficientlyhigher than the temperature used in the assay (65° C.) to minimize theeffect of the melting temperature of probes on ligation efficiency.

All oligonucleotide probes were synthesized using reagents and a 394automated DNA synthesizer from Applied Biosystem Division ofPerkin-Elmer Corporation, Foster City, Calif. Synthesis ofoligonucleotides with a fluorescent dye, 6-FAM (6-carboxy Fluorescin),attached at the 5′ end was done using 6-FAM Amidites from AppliedBiosystem Division of Perkin-Elmer Corporation. The oligonucleotide witha 3′ FAM was made by using a 3′-Amino-modifier C3-CPG column from GlenResearch (Sterling, Va.) for the initial DNA synthesis, and the FAMgroup was then attached through the 3′-amino group using NHS-FAM(N-hydroxyl Succinimide ester of FAM) from the Applied BiosystemDivision of Perkin-Elmer Corporation. All oligonucleotides used in thisstudy were purified by polyacrylamide gel electrophoresis and recoveryof DNA from gel slices using C-18 Sep-Pak Cartridges from WatersDivision of Millipore.

Oligonucleotide probes, RP5′A, RP5′C, RP5′G, RP5′T, and Com3′F were5′-phosphorylated in a solution containing 50 mM Tris-HCl, pH 7.6, 10 mMMgCl₂, 1 mM EDTA, 10 mM DTT, 1 mM ATP, 1 nmole of gel-purifiedoligonucleotides and 10 units of T4 polynucleotide kinase (New EnglandBiolabs, Beverly, Mass.) at 37° C. for 45 minutes. The reaction wasstopped by adding 0.5 μl of 0.5 M EDTA, and the kinase washeat-inactivated by incubation at 64° C. for 20 minutes. Thephosphorylated oligonucleotides were stored at −20° C. in aliquotsbefore use.

The fluorescent fidelity assay of Tth DNA ligase was carried out in 40μl of buffer containing 20 mM Tris-HCl, pH 7.6; 10 mM MgCl₂; 100 mM KCl;10 mM DTT; 1 mM NAD⁺; and 500 fmol (12.5 nM) of nicked duplexsubstrates. DNA probes were denatured by incubating the reaction mixtureat 94° C. for 2 min, and re-annealed at 65° C. for 2 min. Ligations werestarted by the addition of 5 fmol of the thermostable ligase and carriedout at 65° C. 5 μl aliquots were removed at 0 hr, 2 hr, 4 hr, 6 hr, 8hr, and 23 hr, and mixed with 18 μl of a stop solution (83% Formamide,8.3 mM EDTA, and 0.17% Blue Dextran). 5 μl of this mixture was denaturedat 93° C. for 2 min, chilled rapidly on ice prior to loading on an 8 Murea-10% polyacrylamide gel, and electrophoresed on a 373A automated DNASequencer from Applied Biosystem Division of Perkin-Elmer Corporation,Foster City, Calif. Fluorescently labeled ligation products wereanalyzed and quantitated using Genescan 672 software (Applied BiosystemDivision of Perkin-Elmer Corporation, Foster City, Calif.). Genescan 672software provided analyzed data in the form of a gel image and a tablecontaining the peak height and peak area of each peak in each lane.Typically, two bands were seen in each lane representing each reaction.The lower one was the free fluorescent common oligonucleotide, the upperone was the upper strand of the ligation product. The product yield inpercentage was calculated as product over total intial substrates times100. The amount of product was calculated as the peak area of theappropriate ligation product. The amount of initial substrates werecalculated by adding the peak area of the product peak to that of thefree fluorescent oligonucleotide peak. Results were plotted usingDeltaGraph Pro3 software (DeltaPoint, Inc. Monterey, Calif.) with timeas the X-axis and yield as the Y-axis.

Strategy for Testing Wild-type Tth DNA Ligase Fidelity

A fluorescent assay using nicked substrates was developed for testingTth DNA ligase fidelity. The nicked duplex substrate was generated byannealing two adjacent oligonucleotide probes (one of them containing afluorescent dye FAM) to a longer complementary template (bottom) strand(see FIG. 13 for sequences). For clarity, the fluorescently labeledprobe is defined as the common oligonucleotide while the probecontaining the test base at its terminus is called the discriminatingoligonucleotide. A set of 14 oligonucleotides (FIG. 13) were used togenerate all possible combinations of different single-base pair matchesand mismatches at the 3′- and 5′-sides of the nick. Both common anddiscriminating oligonucleotides were designed such that their meltingtemperature was at least 10° C. higher than the assay temperature (65°C.). This presumably minimized the effect of differences inoligonucleotide hybridization on ligation efficiency. The ligation timewas extended to 23 hours allowing for accurate quantification ofmismatch ligation products. Ligation of the two adjacentoligonucleotides formed a longer fluorescent product, which wasseparated on a denaturing polyacrylamide gel and analyzed.

Fidelity of Nick Closure by Wild-type Tth DNA Ligase

A time course for nick closure by Tth DNA ligase using substrates wherethe discriminating base is on the 3′-side of the nick is recorded inTable 3 and is shown in FIG. 15.

TABLE 3 Ligation yield generated by Tth DNA ligase with different DNAsubstrates containing different baseparing on the 3′ side of the nick.Base-pairing 0 hour 2 hour 4 hour 6 hour 8 hour 23 hour A-A 0%  0%  0% 0%  0%  0% A-T 0% 85.3% 88.9% 90.2% 92% 93.2% A-G 0%  0%  0%  0%  0% 0% A-C 0%  0%  0%  0%  0%  1.1% C-A 0%  0%  0%  1.3%  1.8%  2% C-T 0% 0%  0%  0%  0%  0% C-G 0% 82% 88% 90% 96.5% 93.3% C-C 0%  0%  0%  0% 0%  0% G-A 0%  0%  0%  0%  0%  0% G-T 0%  2.5%  3.8%  5.46%  7.5% 11%G-G 0%  0%  0%  0%  0%  0% G-C 0% 82.6% 87.4% 87.4% 88.9% 91.3% T-A 0%83.8% 86.5% 88.4% 89% 92.6% T-T 0%  0%  0%  0%  0%  0% T-G 0%  2.2% 3.8%  4.45%  8.8% 13.4% T-C 0%  0%  0%  0%  0%  0%

Each panel in FIG. 15 shows the yield of product formed with the samediscriminating oligonucleotide and common oligonucleotide annealed tofour template strands which differ by a single base. All perfectlymatched substrates yielded over 80% product within 2 hrs. (FIG. 15). Ofall 12 mismatches tested on the 3′-side of the nick, T-G and G-Tmismatches were less efficiently discriminated, with yields of about 2%after 2 hrs. accumulating to about 15% after 23 hrs. incubation, (FIG.15). These results on discriminating different 3′ side mismatches withTth DNA ligase are similar to those reported for T4 DNA ligase(Landegren, U., et al., Science 241:1077-1080 (1988), which is herebyincorporated by reference), although Tth DNA ligase does not requirehigh salt, spermidine, or very low enzyme concentrations to suppressmismatch ligations (Wu, D. Y., et al., Gene, 76:245-254 (1989) andLandegren, U., et al., Science, 241:1077-1080 (1988), which are herebyincorporated by reference).

When the mismatches were located at the 5′-side of the nick, the enzymestill exhibited stringent discrimination against A-G, C-C, G-G, and T-Cmismatches. The results of this experiment are recorded in Table 4 andare plotted in FIG. 16.

TABLE 4 Ligation yield generated by Tth DNA ligase with different DNAsubstrates containing different baseparing on the 5′ side of the nick.Base-pairing 0 hour 2 hour 4 hour 6 hour 8 hour 23 hour A-A 0%  3.2% 4.45%  6.7%  7.45% 23.6% A-T 0% 79.6% 82.2% 84.2% 85.6% 89.1% A-G 0% 0%  0%  0%  0%  0% A-C 0%  2.8%  4.4%  7.4%  8.7% 22.8% C-A 0%  7.3%13.5% 19% 23.7% 38.2% C-T 0%  0%  1.47%  2%  2.7%  6.5% C-G 0% 78% 83.5%84.8% 84.2% 89.5% C-C 0%  0%  0%  0%  0%  0.86% G-A 0%  1.8%  4.4%  6.8%10.5% 20% G-T 0%  2.1%  5.34%  7.5% 11.7% 20.5% G-G 0%  0%  0%  0%  0% 1.03% G-C 0% 76.2% 80.9% 84.1% 86.3% 87.2% T-A 0% 78.7% 83.8% 84.5%86.4% 87.8% T-T 0% 14.3% 22.7% 30% 38.6% 44.5% T-G 0%  8.9% 14.3% 20.8%30.4% 44.9% T-C 0%  0%  0%  0%  0%  1.1%

Ligation yields of mismatches A-C, A-A, C-A, G-A and T-T, were barelydetectable after extended incubation (23 hrs.) when placed at the3′-side of the nick, but became quite significant when placed at the5′-side of the nick. Different ligation rates observed between isostericmismatched substrates, G-A and A-G, or C-T and T-C, suggest that theserates are influenced by other factors, possibly stacking interactionswith neighboring bases. Overall, these results indicate that Tth DNAligase discriminates all mismatches at the 3′-side of the nick moreefficiently than mismatches at the 5′-side of the nick.

If ligation fidelity were mainly dependent on the cumulative stabilityof base pairing near the junction, the internal stability would havebeen predicted to be higher for the DNA sequence at the 5′-side of thenick than at the 3′-side of the nick. The internal stability iscalculated as the sum of the free energy of five continuous bases, andwas found to be lower on the 5′-side of the nick (calculated using theOligo 4.0 program from National Biosciences Inc., Plymouth, Minn.).Therefore, the observed higher fidelity to mismatches on the 3′-side ofthe nick by Tth DNA ligase was not caused by a specific sequence withinthe discriminating oligonucleotides, but by specific requirements of thenick structure recognized by this enzyme.

Improved discrimination of mismatches located at the 3′-side of the nickcompared to those at the 5′-side of the nick was reported for A-A andT-T mismatches using bacteriophage T4 DNA ligase (Wu, D. Y., et al.,Gene, 76:245-254 (1989), which is hereby incorporated by reference).This difference was attributed to either (i) a single-base mismatchwhich destablized annealing of the octamer probe used on the 3′-sidemore than the tetradecamer probe used on the 5′ side, or (ii) anintrinsic feature of T4 DNA ligase (Wu, D. Y., et al., Gene, 76:245-254(1989), which is hereby incorporated by reference). These resultssupport the second hypothesis, because the oligonucleotides used in thisassay were similar in length and melting temperature. A detailedanalysis of the Vaccinia virus ligase showed that all mismatches on the5′-side were ligated more efficiently than mismatches on the 3′-side(Shuman, S., Biochem., 34:16138-16147 (1995), which is herebyincorporated by reference). Similar results have also been shown for DNAligase from Saccharomyces cerevisiae although the mismatches tested onthe 5′-side of the nick were not the same as those at the 3′-side of thenick (Tomkinson, A. E., et al., Biochemistry, 31:11762-11771 (1992),which is hereby incorporated by reference). Also, the AP sites at the3′-side of nicks were less efficiently ligated by T4 DNA ligase comparedto the AP sites at the 5′-side of nicks (Goffin, C., et al., NucleicAcids Res., 15(21):8755-8771 (1987), which is hereby incorporated byreference). Therefore, the more stringent requirement for the canonicalstructure at the 3′-side of the nick compared to the 5′-side of the nickmay be general to all DNA ligases.

The lower fidelity against T-G or G-T mismatches on the 3′-side by Tthand T4 DNA ligases mirrors the fidelity of DNA polymerases. The mostcommon mispairs formed by insertion errors of DNA polymerase involve Gpairing with T, although there were substantial variations observeddepending on the DNA polymerase, the method of assay, and the specificsite investigated (Echols, H., et al., Annu. Rev. Biochem., 60:477-511(1991), which is hereby incorporated by reference). It was also shownthat G-T, A-C, and G-A mispairings were the most frequent ones allowedby the E. coli pol III both in vivo (Schaaper, R. M., Proc. Natl. Acad.Sci. USA, 85:8126-8130 (1988), which is hereby incorporated byreference) and in vitro (Sloane, D. L., et al., Nucleic Acids Res.,16:6465-6475 (1988), which is hereby incorporated by reference).Furthermore, Taq DNA polymerase extension of a 3′ mismatched primer ismore efficient for T-G and G-T mismatches than other mismatches at lowdNTP concentrations (Kwok, S., et al., Nucleic Acids Res., 18:999-1005(1990), which is hereby incorporated by reference). NMR and X-raycrystallography analysis of the structure of aberrant base pairs induplex DNA oligonucleotide have indicated that G-T, A-C, and G-A pairsexisted as “wobble” structures which differs slightly in dimensions froma normal Watson-Crick base pair (Hunter, W. N., et al., Nature,320:552-555 (1986) and Patel, D. J., et al., Fed. Proc. Fed. Am. Soc.Exp. Biol., 43:2663-2670 (1984), which are hereby incorporated byreference). It was suggested that a well-stacked wobble pair may resultin a more stable structure than a poorly stacked base pair approximatingWatson-Crick geometry more closely. Both bacterial and mammalian DNAligases have a very high fidelity against all purine-purine mismatches,including G-A or A-G mismatches on the 3′-side (Landegren, U., et al.,Science, 241:1077-1080 (1988); Husain, I., et al., J. Biol. Chem.,270:9683-9690 (1995); and Shuman, S., Biochem., 34:16138-16147 (1995),which are hereby incorporated by reference), suggesting that base pairstability is not the predominant factor influencing ligase fidelity.Furthermore, the related Vaccinia virus ligase and mammalian DNA ligaseIII both demonstrate even lower fidelity for C-T mismatches than G-Tmismatches (Husain, I., et al., J. Biol. Chem., 270:9683-9690 (1995);and Shuman, S., Biochem., 34:16138-16147 (1995), which are herebyincorporated by reference), while the Tth and T4 DNA ligases have alower fidelity for G-T mismatches (Landegren, U., et al., Science,241:1077-1080 (1988), which is hereby incorporated by reference, withregard to T4 ligase), with the Tth DNA ligase having virtually nomis-ligations for C-T mismatches on the 3′-side. Thus, the fidelity ofligases is influenced not only by the compromise between increasedstability and decreased helix distortion of a mismatched base pair, butalso by specific structural determinants of the individual ligaseprotein.

Improving the Fidelity of Tth DNA Ligase

The fidelity of T4 DNA ligase has been expressed as a specificity ratio;defined as the ratio of ligation product formed in the presence ofmatched versus mismatch template in the presence of 100 nM of templateat 30° C. with one unit of enzyme (Wu, D. Y., et al., Gene, 76:245-254(1989), which is hereby incorporated by reference). At high saltconcentrations (200 mM), the specificity ratio increased from 6 to 60for T-T mismatches and from 1.5 to 40 for A-A mismatches. These morestringent conditions also increased the K_(m) 4 fold and decreased theV_(max) approximately 30 fold (Wu, D. Y., et al., Gene, 76:245-254(1989), which is hereby incorporated by reference). The fidelity of S.cerevisiae DNA ligase I was defined as the ligation efficiency ofsubstrates containing perfect matches vs. mismatches in the presence of3 to 900 ng of enzyme at 20° C. for 30 min (Tomkinson, A. E., et al.,Biochemistry, 31:11762-11771(1992), which is hereby incorporated byreference). The efficiency of this DNA ligase was found to decrease 50fold when a T-G mismatch was present on the 3′-side of the nick. Theligation efficiency decreased by greater than 100-fold when a C-Tmismatch was present on the 3′-side of the nick and a T-C mismatch onthe 5′ side of the nick in the same substrate. It is difficult tocompare the fidelity of different DNA ligases using either of the twoterms described above, because they will vary when different incubationtimes and substrates are used.

In order to standardize comparisons with different substrates andenzymes, the fidelity of Tth DNA ligase is defined as a ratio of theinitial rate of perfect match ligation over the initial rate of mismatchligation. Among substrates containing a mismatch on the 3′-side of thenick, T-G or G-T mismatches are the most difficult to discriminate, andwere therefore used as a standard assay for determining the fidelityratio of Tth DNA ligase. Initial rates of C-G perfect-match ligationover T-G mismatch ligation at 65° C. gave a fidelity ratio of 4.5×10² byTth DNA ligase (Row 1, FIG. 18). Use of shorter discriminating probes(T_(m) approximately 65° C.) did not dramatically change perfect-matchligations, but did decrease mismatch ligation. Thus, destabilizing themismatched probe improved the fidelity ratio about 3-fold to 1.5×10³(Row 2, FIG. 18). This Tth DNA ligase fidelity ratio is at least 24 foldhigher than reported for T4 ligase (Wu, D. Y., et al., Gene, 76:245-254(1989), which is hereby incorporated by reference), on a T-T mismatchwhich is far easier to discriminate than a G-T mismatch used herein, seeFIG. 15).

This exquisite specificity of Tth DNA ligase has been exploited inligase chain reaction (i.e. LCR) to detect a mutant allele (A-T match)in the presence of a 200-fold molar excess of wild-type sequence (G-Tmismatch) (Balles, J., et al., Mol. Gen. Genet., 245:73440 (1994), whichis hereby incorporated by reference). Several medical DNA detectionproblems will require an even greater specificity. Two approaches toimprove further the fidelity ratio of Tth DNA ligase by destabilizingthe enzyme-substrate interactions have been devised as described below.

Improving the Fidelity of Tth DNA Ligase by Modifying the DNA Substrate

An elegant method for improving allele-specific PCR is based on usingprimers with a deliberate mismatch adjacent to the discriminating 3′base (Cha, R. S., et al., PCR Methods and Applications, 2:14-20 (1992)and Rust, S., et al., Nucleic Acids Res., 21(16):3623-3629 (1993), whichare hereby incorporated by reference). This destabilizing mismatch didnot dramatically reduce Taq polymerase extension of the correct targetallele, but owing to a double mismatch of the other allele, theextension efficiency of the incorrect allele was reduced by a factor of100 to 1000-fold (Cha, R. S., et al., PCR Methods and Applications,2:14-20 (1992), which is hereby incorporated by reference).

This approach was adapted for use in ligation reactions by deliberatelyintroducing an A-C mismatch at the third position on the 3′-side of thenick. (Shown as bold letters in both perfect match and mismatch DNAsubstrates, FIG. 18, Row 3). The original perfect match substrate nowhas a single mismatch, and the original mismatch substrate now has twomismatches (one right on the 3′-side of the nick; the other, three basesin on the 3′-side of the same probe). This A-C mismatch in the thirdposition reduced the ligation efficiency of a 3′ matched (C-G) substratealmost 10 fold with 5 fmol Tth DNA ligase (data not shown). In order toobtain an initial rate comparable to that with normal perfect matchligations, the amount of enzyme was increased to 50 fmol. The ligasefidelity ratio increased by about 4-fold (to 5.8×10³) when the extramismatch was introduced (FIG. 18, Row 3). The internal mismatch has agreater destabilizing effect on the structure near the nick for theprobe containing the 3′ mismatch at the nick, than the probe containingthe perfect match at the nick. Therefore, the overall fidelity of theligase was improved. Similar results (4-fold increase) were alsoobtained when a T-G or G-T mismatch was introduced at the same thirdposition on the 3′-side of the nick (data not shown). If the extramismatch (C-A) was introduced into the second position on the 3′ side ofthe nick, the ligation of perfect match at the 3′-side of the nick byTth DNA ligase was strongly inhibited (175-fold lower than with nomismatch at the second position). In contrast, Taq DNA polymerase wasnot affected by the extra mismatch at the second position from the3′-end of the primer (Cha, R. S., et al., PCR Methods and Applications,2:14-20 (1992), which is hereby incorporated by reference). A keydifference between PCR and LCR is that an adjacent mismatch only affectsextension from the target during the initial PCR cycle, whereas, in LCR,ligation on the target and products is affected during every cycle ofthe LCR reaction.

As an alternative to a mismatched base pair, a universal nucleotideanalogue might maintain DNA helix integrity with a perfect matchsubstrate while still destabilizing a nearby mismatch, and thus furtherimprove ligation fidelity. Oligonucleotides containing the nucleotideanalogue 3-nitropyrrole deoxyribonucleotide (Q) at multiple sites inplace of the natural nucleotides have been shown to function effectivelyas sequencing and PCR primers (Bergstrom, D. E., et al., JACS,117:1201-1209 (1995) and Nichols, R., et al., Nature, 369:492-493(1994), which are hereby incorporated by reference). 3-Nitropyrrolepresumably allows preservation of helix integrity, because it issufficiently small to fit opposite any of the four natural bases and hashigh stacking potential due to a highly polarized π-electronicconfiguration. T_(m) studies on Q containing oligonucleotides indicatethat Q base pairs with relatively little discrimination (ΔT_(m)=3° C.)but the stability of Q-A (most stable), Q-T, Q-C, and Q-G (least stable)base pairs is significantly less than that of an A-T or C-G base pair.Consequently, Q, if located three nucleotides in from the 3′-end of aprobe could significantly enhance local melting if it were present inconjunction with a mismatch at the 3′-position, while at the same timepreserving helix integrity more than a mismatch when present inconjunction with a base pair match at the 3′-end. When sequencesSLP3′QTC and SLP3′QTT were tested as ligation substrates for Tth DNAligase, an even better fidelity ratio (9-fold increase) was obtained(FIG. 18, Row 4). Note that the base on the template strand was changedfrom C to A. As determined by the initial rates of perfect matchligation, the Q base analogue appears to pair to A and T equally well,less well to C, and very poorly with G. The ratio of initial ratesobtained when Q was paired with different bases is T:A:C:G=23:16:5:1. Asobserved with C/A mismatches above, when the Q analogue is located atthe second position, the ligation rate is also strongly inhibited (55fold lower than with no analogue at the second position).

On the basis of modeling studies (QUANTA/CHARMM), Q can fit oppositeboth T and A with minimal perturbation of the helix structure(Bergstrom, D. E., et al., JACS, 117:1201-1209 (1995), which is herebyincorporated by reference). In one case, Q would assume the anticonformation (Q-T), and in the other a syn conformation (Q-A).Nevertheless, studies in progress suggest that hydrogen bonding playsonly a minor role in 3-nitropyrrole-natural base interactions.

Improving the Fidelity of Tth DNA Ligase by Site-directed Mutagenesis ofthe Ligase Protein

The fidelity of DNA polymerases may be decreased by site-directedmutagenesis of motifs associated with primer-template binding (HIVpolymerase) (Beard, W. A., et al., J. Biol. Chem., 269:28091-28097(1994), which is hereby incorporated by reference) or the exoIII motifof Phi29 DNA polymerase (Soengas, M. D., et al., The EMBO J.,11(11):4227-4237 (1992), which is hereby incorporated by reference), T4DNA polymerase (Reha-Krantz, L. J., et al., J. Virol., 67(1):60-66(1993) and Reha-Krantz, L. J., et al., J. Biol. Chem., 269:5635-5643(1994), which are hereby incorporated by reference), or human DNApolymerase alpha (Dong, Q., et al., J. Biol. Chem., 268:24163-24174(1993), which is hereby incorporated by reference). Occasionally, thissame exoIII motif or motif “A” yields increased fidelity mutants alsoknown as “antimutator” strains, which reflects the complex interplaybetween the polymerizing and 3′-5′ exonuclease activities of theseenzymes in modulating overall fidelity (Reha-Krantz, L. J., et al., J.Virol., 67(1):60-66 (1993); Reha-Krantz, L. J., et al., J. Biol. Chem.,269:5635-5643 (1994); Dong, Q., et al., J. Biol. Chem., 268:24175-24182(1993); and Copeland, W. C., et al., J. Biol. Chem., 268:11041-11049(1993), which are hereby incorporated by reference).

Mutant Tth DNA ligases, which retained near wild-type nick-closingactivity, were assayed for changes in fidelity. Two mutant ligases,K294R and K294P were shown to have increased their fidelity ratios (FIG.18, Rows 5-8). With regular substrates, the fidelity increased about4-fold by K294R and 11-fold with K294P. When Q base analogues were usedtogether with mutant Tth DNA ligases, the fidelity of the ligationreactions were increased by almost 20-fold (K294R and K294P), althoughhigher concentrations of mutant ligase were required. In subsequentexperiments (see Table 5 below), K294Q and K294L were also shown to haveincreased fidelity ratios.

In summary, a quantitative fluorescence assay has been developed foranalyzing the fidelity of Tth DNA ligase. This enzyme exhibitssignificantly greater discrimination against all single-base mismatchesat the 3′-side of the nick by comparison to those at the 5′-side of thenick. Among all twelve possible single-base pair mismatches at the3′-side of the nick, only T-G and G-T mismatches generated aquantifiable level of ligation products after extended incubation. Thefidelity of Tth DNA ligase can be improved further by designingdiscriminating oligonucleotides with melting temperature values near theligation temperature of 65° C., by introducing a deliberate mismatchbase or a nucleotide analogue into the left third position on the3′-side of the nick, and/or by using mutant Tth DNA ligase.

Numerous medical problems will require exquisite single-basediscrimination, for example, finding rare cancer cells among many normalcells, distinguishing pathogenic micro-organisms among normal flora,identifying oncogenic HPV strains in mixed infections, and detecting theemergence of drug resistant organisms. While these model studies werelimited to either pure mismatched or matched target substrates,nucleotide analogues and mutant Tth DNA ligases have recently been usedto improve discrimination of a minor correct target in a mixture ofincorrect target. This more closely mimics DNA detection in biologicalsamples.

Example 16

Oligonucleotide Synthesis and Purification

All oligonucleotides were synthesized on an ABI 394 DNA Synthesizer(Applied Biosystems Inc., Foster City, Calif.). For oligonucleotideslabeled with FAM (i.e. Com 610 3′F), a fluorescein CPG (Glen Research)column was used to introduce a fluorescein molecule to the 3′-terminusof the oligonucleotide. Cleavage of the oligonucleotide from the supportwith 30% (Wt/Vol) NH₄OH required 2 hr. at room temperature. Theoligonucleotide was subsequently deprotected at 55° C. for 4 hr. Allother oligonucleotides used in LDR were purified by polyacrylamide gelelectrophoresis on 10% acrylamide/7M urea gels. Oligonucleotides werevisualized after electrophoresis by UV shadowing against a lightiningscreen and excised from the gel (Applied Biosystems Inc., 1992). Theywere then eluted overnight at 64° C. in TNE buffer (100 mM Tris/HCl pH8.0 containing 500 mM NaCl and 5 mM EDTA) and recovered from the eluateusing Sep Pak cartridges (Millipore Corp, Milford, Mass.) following themanufactures instructions.

Oligonucleotides were resuspended in 100 μl TE. Typical concentration ofa stock solution is about 1 mM. For LDR, gel purified stock solutionswere about 100 μM=100 pmoles/μl.

Example 17

Chemical Phosphorylation of Oligo Com 610-3′F

The downstream, common oligonucleotide was phosphorylated at the 5′ endusing a chemical phosphorylation reagent (Glen Research). The use ofthis reagent is an alternative to the enzymatic techniques foroligonucleotide phosphorylation, with the advantage of allowingphosphorylation efficiency to be determined. Chemical phosphorylationreagent has proved to be a fast and convenient method forphosphorylation at the 5′-terminus of oligonucleotides. The compound isstored desiccated at 4° C. For synthesis on the ABI394 machine, a 0.1Msolution is prepared by adding 1 ml of anhydrous acetonitrile per 100μmoles of the compound. The liquid is allowed to sit for approximately 5minutes, swirling occasionally to ensure complete dissolution. Synthesisof the oligonucleotide was performed with modifications to the synthesiscycle according to the manufacturer's instructions. For example, the DMTgroup was removed on the synthesizer by the standard deblocking methodto determine the coupling efficiency.

Quantitative Detection of Target Containing a Single-base Mutation (T:GMismatch), in the Presence of an Excess of Normal Template, UsingWild-type and Mutant Tth DNA Ligases

An assay has been developed which can quantify the amount of a lowabundance sequence (cancer mutation) in an excess of normal DNA. Usingwild-type and mutant Tth DNA ligases in an LDR assay, the fraction of“cancer” DNA in a mixture of normal and cancer DNA could be quantified.The probes used to determine the limits of detection are shown in FIG.19, with sequences in FIG. 20. Two oligonucleotides were hybridized tothe target such that the 3′ end of the upstream probe (SLP3′TTT) isimmediately adjacent to the 5′ end of the downstream probe (Com610-3′F). Tth DNA ligase can then join the two adjacentoligonucleotides, provided that the nucleotides at the junction arecorrectly base-paired with the target strand. The probes used in thisreaction create a T:G mismatch on the “Normal” template and an T:A matchon the “Cancer” template at the ligation junction. LDR experiments weredone in triplicate using 12.5 nM (250 fmole) of the T:G mismatchedtemplate (“Normal”; GLg.m3A and Glg.m3Arev), containing from 0 to 2.5 nM(50 fmole) of perfect matched template (“Cancer”; ALg.m3A andAlg.m3Arev) in the presence of 25 fmol of purified wild-type and mutantenzyme K294R (See FIG. 21). Products are separated on an ABI 373A DNAsequencing apparatus and fluorescent signal quantified. If yield isdefined as Product/(Product+Probe), the yields are artificially high dueto quenching of the overloaded probe signal. Therefore, a dilutedproduct control was run to generate a calibration number (1 fmole=600peak area units). The amount of product in a given sample in fmol wascalculated by dividing the total peak area of product by 600. Thismethod will underestimate product formation if less than the full 5 μlligation mix is loaded per lane. Both enzymes generated about the sameamount of product, but the mutant K294R enzyme gave less backgroundmismatch ligation. Therefore, the signal-to-noise ratio was almostdouble for the mutant enzyme compared to the wild-type ligase (See FIG.22). With the K294R mutant enzyme, the signal-to-noise ratio was 3.3 fordistinguishing one “Cancer” template in 500 “Normal” template, andincreased to 5.4 for distinguishing one in 250 “Normal” templates (Seenumbers in FIG. 22, as well as a similar experiment described in Table5). This assay compares the ability of ligase to discriminate the mostdifficult case; a T:G mismatch from an T:A perfect match.

The results shown in FIGS. 21 and 22 demonstrate the ability of Tth DNAligase to distinguish the presence of a perfect match substrate(“Cancer” mutation) in the presence of an excess of mismatch substrate(“Normal”). To quantify precisely the amount of “cancer” template, it isnecessary to determine the amount of “normal” template present, whichmay be done in two ways. The simplest method is to include a small ratioof fluorescently labeled probe in the original PCR mix, and quantify theamount of product generated using capillary electrophoresis or anautomated DNA sequencer. The second approach uses the quantitativenature of ligation reactions to determine the original productconcentration. FIG. 23 shows the amount of LDR product generated from0.005 nM (0.1 fmol), to 0.5 nM (10 fmol) “Normal” template (GLg.m3A andGlg.m3Arev) using probes that are perfectly matched at the junction(SLP3′TTC and Com 610-3′F). With the K294R mutant enzyme, thesignal-to-noise ratio was 4.5 for detecting 0.005 nM (0.1 fmol) “Normal”template and improved to 7.4 for detecting 0.0125 nM (0.25 fmol)“Normal” template (data not shown). By comparing the amount of LDRproduct from an unknown sample with the calibration curve, the total DNAin a sample can be quantified. This can be achieved by directly testingundiluted samples of amounts from 100 attomoles (0.005 nM) to 1 0femtomoles (0.5 nM), or testing dilutions when working with higherquantities/concentrations of DNA. These results demonstrate that theamount of specific DNA present in an unknown sample can be determined,as well as the percentage (amount) containing a single-base (“Cancer”)mutation.

Detection of Target Containing a Single-base Mutation in the Presence ofan Excess of Normal Template Using Wild-type and Mutant Tth DNA Ligases

To explore further the use of wild-type and mutant Tth DNA ligases incancer detection, the ligation detection reaction was optimized in acompetition assay which more closely mimics the biological problem ofdistinguishing a cancer cell in an excess of normal cells. This type ofassay requires not only that the ligase seal the correct sequence, butthat it must do so when most of the enzyme is bound to the incorrectsubstrate. Therefore, higher concentrations of enzyme are required thanin the previous assay, and these higher concentrations may lead toincreased background (mismatched) ligations. Two oligonucleotides (SLP3′TTT and Com 610 3′F) are permitted to hybridize to the denaturedtarget such that the 3′ end of one oligonucleotide is adjacent to the 5′end of a fluorescent labeled oligonucleotide (see FIG. 19). The ligasecan then join the two adjacent oligonucleotides, provided that thenucleotides at the junction are correctly base-paired with the targetstrand. Initial experiments on LDR were done in triplicate using themismatched template (“Normal”), and perfect match template (“Cancer”)independently, or in combination with each other in the presence of 25fmol of purified wild type and mutant enzymes K294R and K294L (Table 5).

TABLE 5 Amount of Product Formed (fmol) with Different Templates Tth DNALigase 10 fmol C Template in 250 2.5 fmol of C Template in Conc 250 fmolN 10 fmol C fmol of N Template 250 fmol of N Template (1.25 nM) TemplateTemplate (1:25) (1:100) Wild-Type 1.45 32.5 11.2 4.9 Mutant 0.56 33.212.9 3.3 K294R Mutant ND 29.6 9.7 3.4 K294L Signal/ — — 7.7 3.4 NoiseRatio Wild-Type Mutant — — 23.1 5.9 K294R Table 5. Comparison of thewild type and mutant Tth ligase in detecting the Cancer signal in thepresence of 25 and 100 fold excess of the normal template. N = Normaltemplate; GLg.m3A and GLg.m3A rev; C = Cancer template; ALg.m3A andALg.m3A rev. The four different conditions used in a ligation detectionreaction was as follows i) 12.5 nM (250 fmol) of Normal (mismatch)Template ii) 0.5 nM (10 fmol) of Cancer (perfect match) Template iii)0.5 nM (10 fmol) of Cancer Template # in 12.5 nM (250 fmol) of NormalTemplate and iv) 0.125 nM (2.5 fmol) of Cancer Template in 12.5 nM (250fmol) of Normal Template. The probes used in this reaction create a T:Gmismatch on the “Normal” template and a T:A match on the “Cancer”template at the ligation junction. Each reaction was carried out in a 20μl volume of a reaction buffer containing 25 nM (500 fmol) of each ofthe two probes (SLP3′TTT and Com 610 3′F), and 1.25 (25 fmol), of eitherthe # wild-type or mutant enzyme. Data was analyzed using the Genescan672 software. In calculating the signal to noise ratios, the signal wasdefined as the amount of perfect match product formed in the presence of25- or 100-fold N template, the value of noise was taken as the amountof mismatch product formed with 12.5 nM (250 fmol) N template. ND = notdetectable.

(The K294P mutation causes loss of thermostability and, therefore, wasnot used in further studies). The amount of LDR product generated (0.56fmol) by the mutant K294R on the mismatched template, was reduced bymore than 2-fold as compared to the wild-type enzyme (1.45 fmol). LDRwas also performed with the perfect match and mismatch templatestogether in a ratio of 1:25 and 1:100, respectively. For both wild-typeand mutant enzymes, the product generated in the presence of themismatch template was less than the product generated by the perfectmatch template alone. The use of K294R results in a 1.75 to 3 foldhigher signal-to-noise ratio. Since no background was detected with theK294L mutant, a signal-to-noise ratio could not be calculated. Thus,these results support the finding that the mutant K294R exhibits ahigher fidelity in discriminating a mismatch over the wild-type enzyme.This increased fidelity is probably due to a change in the specificityconstant of this mutant thermostable enzyme. The specificity of anenzymatic reaction is determined by the catalytic constant, k_(cat), andthe apparent binding constant, K_(M), and expressed as the specificityconstant k_(cat)/K_(M). Any modifications made on the enzyme itself,substrate, or reaction conditions, which affect k_(cat) or K_(M) orboth, will change the specificity. The use of a mutant enzyme mayinfluence the stability of the perfect matched and mismatched enzyme-DNAcomplexes to a different extent, so that discrete K_(M) effects areexerted on these ligation reactions. In a competitive reaction, such asligation of perfectly matched and mismatched substrates, the ratio ofthe specificity constant may be altered as a consequence of K_(M), andpossible k_(cat) changes for each substrate. All mutant enzymes whichsatisfy the equation below (shown for K294R) will give increaseddiscrimination of cancer-associated mutations in the presence of anexcess of normal DNA.$\frac{\left\lbrack {k_{cat}/K_{M}} \right\rbrack_{{K294R},{match}}}{\left\lbrack {k_{cat}/K_{M}} \right\rbrack_{{K294R},{mismatch}}} > \frac{\left\lbrack {k_{cat}/K_{M}} \right\rbrack_{{Wt},{match}}}{\left\lbrack {k_{cat}/K_{M}} \right\rbrack_{{Wt},{mismatch}}}$

Alternatively, the second aspect of the present invention can beexpressed in terms of a fidelity ratio (i.e. the initial rate ofligating a substrate with a perfect match at the 3′ end divided by theinitial rate of ligating a substrate with a mismatch at the 3′ end) asfollows:${\frac{\left\lbrack k_{1} \right\rbrack_{{K294R},{match}}}{\left\lbrack k_{1} \right\rbrack_{{K294R},{mismatch}}} > \frac{\left\lbrack k_{1} \right\rbrack_{{Wt},{match}}}{\left\lbrack k_{1} \right\rbrack_{{Wt},{mismatch}}}} = {{Fidelity}\quad {ratio}}$

In the above equation, [k₁]_(match) represents the initial rate constantfor ligating the first and second oligonucleotide probes hybridized to atarget nucleotide sequence with a perfect match at the ligation junctionbetween the target nucleotide sequence and the oligonucleotide probehaving its 3′ end at the ligation junction. [k₁]_(mismatch) representsthe initial rate constant for ligating the first and secondoligonucleotide probes hybridized to a target with a mismatch at theligation junction between the target nucleotide sequence and theoligonucleotide probe having its 3′ end at the ligation junction. Forthe mutant thermostable ligase, [k₁]_(match) divided by [k₁]_(mismatch)(=fidelity ratio) is greater than the fidelity ratio for wild-typeligase. All mutant enzymes which satisfy the equation above (shown forK294R) will give increased discrimination of cancer-associated mutationsin the presence of an excess of normal DNA.

Detection of Target Containing a Single-base Mutation in the Presence ofan Excess of Normal Template Using Nucleotide Analogue Containing Probes

In an effort further to increase ligase fidelity, probes weresynthesized containing the Q₂ or Q₁₈(1-(2′-deoxy-β-D-ribofuranosyl)pyrrole-3-carboxamide) nucleotideanalogues in the 3rd position from the 3′ end of the discriminatingprobe (see FIG. 24). In these experiments, the template base oppositethe probe analogue was either A or T, to allow assaying of the morefavored Q₂:A, Q₂:T, and Q₁₈:T pairings. Using these analogue probes,products generated from the mismatch ligation were reduced approximatelyby 2 to 3-fold as compared to the regular primers (Table 6).

FIG. 24 shows nucleotide analogue containing probes used for assayingligase fidelity. Four different conditions were used to assess thefidelity of the wild-type and mutant Tth DNA ligase in a typical LDRassay as shown in Table 6. Each reaction was carried out in a 20 μlmixture containing 20 mM Tris-HCl, pH 7.6; 1 0 mM MgCl₂; 100 mM KCl; 10mM DTT; 1 mM NAD⁺; 25 nM (500 fmol) of the two short detecting probesand 12.5 nM (250 fmol) of the normal template when used alone, or 125 nM(2.5 fmol), and 0.5 nM (10 fmol), of the cancer template when usedtogether with the normal template in a ratio of 1:100, and 1:25,respectively. The probes used in this reaction create a T:G mismatch onthe “Normal” template and an T:A match on the “Cancer” template at theligation junction. In addition, probe SLP3′QTT creates a Q₂:A or Q₁₈:Tpairing at the 3rd position from the 3′ end. The reaction mixture washeated in a GeneAmp 9600 thermocycler (Perkin Elmer) for 1.5 sec. at 94°C. before adding 25 fmol of the wild-type Tth DNA ligase. Afterincubation with the enzymes for another 30 sec, LDR reactions were runfor 15 sec at 94° C., and 4 min. at 65° C. per cycle for 20 cycles.Reactions were completely stopped by chilling the tubes in anethanol-dry ice bath, and adding 0.5 μl of 0.5 mM EDTA. 2.5 μl ofreaction was mixed with 2.5 μl of loading buffer (83% Formamide, 8.3 mMEDTA, and 0.17% Blue Dextran) and 0.5 μl Gene Scan Rox-1000 molecularweight marker, denatured at 94° C. for 2 min, chilled rapidly on iceprior to loading on an 8 M urea-10% polyacrylamide gel, andelectrophoresed on an ABI 373 DNA sequencer. Fluorescent labeledligation products were analyzed and quantified using the ABI Gene Scan672 software.

FIGS. 25A-D show different forms of oligonucleotide probes withnucleotide analogues for the LDR phase of the PCR/LDR process of thepresent invention. In FIG. 25A, one oligonucleotide probe hybridized toa target nucleotide sequence where the probe has the discriminating baseat its 3′ end (i.e. at the ligation junction) and a nucleotide analogueat the third position from the ligation junction. Such anoligonucleotide probe is a bit unstable but will still undergo ligation,because its 3′ end is complementary to the target nucleotide sequence towhich it is hybridized. FIG. 25B is similar to FIG. 25A except that noligation occurs, because its 3′ end is not complementary to the targetnucleotide sequence to which it is hybridized. In FIG. 25C, theoligonucleotide probe hybridized to the target nucleotide sequence has adiscriminating base at its 3′ end (i.e. at the ligation junction) aswell as at the 2 adjacent positions. In the positions 4, 5, and 6 basesfrom the ligation junction, there are nucleotide analogues. As a result,in FIG. 25C, although the presence of the 3 oligonucleotide analoguesmakes the oligonucleotide probe unstable, ligation will still occur,because there is complete complementarity at the 3 positions closest tothe ligation junction. On the other hand, where there is a mismatch atone or more of the 3 positions closest to the ligation junction, asshown in FIG. 25D, no ligation will occur. Comparing the oligonucleotideprobes used in the procedure of FIGS. 25A-B to those of FIGS. 25C-D, itis apparent that the former has a potential zone of instability at the 3positions closest to ligation junction, while the latter has a potentialzone of instability at the 6 positions closest to ligation junction. Theuse of a 6 position zone of instability instead of a 3 position zone ofinstability has the potential to improve LDR fidelity, because such alarger zone of instability reduces the likelihood that a ligationproduct will form despite the existence of a mismatch at the ligationjunction.

TABLE 6 Amount of Product Formed (fmol) with Different Templates 10 fmolof C 2.5 fmol of C Template in 250 Template in Nucleotide Wild type Tth.250 fmol N 10 fmol C fmol of N 250 fmol of N Signal/Noise Signal/NoiseAnalogue ligase (nM) Template Template Template Template (1:25) (1:100)Q₂:A 1.25 0.56 40.6 11.4 3.4 20.4 6.0 2.5 0.72 51.6 15.0 4.7 20.8 6.65.0 1.40 57.9 20.9 6.5 14.9 4.6 10.0 2.51 62.9 31.0 10.2 12.4 4.1 Q₂:T1.25 N.D. 12.9 5.2 1.5 — — 2.5 N.D. 23.5 8.6 2.8 — — 5.0 0.61 35.9 12.44.6 20.4 7.5 10.0 0.78 38.1 21.2 6.9 27.1 8.9 Q₁₈:T 1.25 0.86 43.2 13.43.7 15.6 4.4 2.5 0.74 28.4 10.1 3.9 13.7 5.3 5.0 0.82 30.1 14.5 5.3 17.76.4 10.0 1.05 35.3 15.8 4.6 15.1 4.3 Regular 1.25 1.45 32.5 11.2 4.9 7.73.4 Probes 3′TTT+61 03′F Table 6 shows a comparison of the Q Analogueswith the regular probes in detecting a “Cancer” signal using increasingconcentrations of the wild-type Tth DNA ligase. N = Normal template(GLg.m3A and GLg.m3A rev) C = Cancer template (Alg.m3A and ALg.m3A rev.)for the Q₂:A assays. N = Normal template (GLg.m3T and GLg.m3T rev) C =Cancer template (ALg.m3T and ALg.m3T rev.) for the Q₂:T and the Q₁₈:Tassays. N.D.; Not Detected. The probes used # in this reaction create aT:G mismatch on the “Normal” template and an T:A match on the “Cancer”template at the ligation junction. In addition, probe SLP3′QTT creates aQ₂:A or Q₁₈:T pairing at the 3rd position from the 3′ end. (See FIG. 24for probe layout). The four different conditions used in the LDR assayunder thermal cycling conditions were the same as described in Table 5,however in this experiment, two Q analogues; Q₂ and Q₁₈, # were used todetermine the amount of product formed with varying concentrations ofthe wild type enzyme ranging from 1.25 nM (25 fmol) to 10 nM (200 fmol).As a control for this experiment, the amount of product formed with 1.25(nM (25 fmol) of the wild type enzyme and 25 nM (500 fmol) of theregular probes was also examined. Columns 3 to 6 display the amount ofproduct formed when different concentrations of the enzyme was used with25 nM (500 fmol) of Q₂, Q₁₈ and regular # probes, respectively. Thesignal to noise ratio for each of these variables are shown in the lasttwo columns.

However, the amount of signal generated from the perfect match ligationsremained relatively constant. Higher enzyme concentration (for example,10 nM (200 fmol)) leads to the saturation of the perfect match templatewith the nucleotide analogues, so that more enzyme is free to bind tothe mismatch template. As a consequence, higher mismatch signals wereobserved with increased enzyme concentrations. In the presence ofperfect match and mismatch templates (1:25 and 1:100), the absolutesignals generated from the perfect match was less than that generated bythe perfect match alone, for both unmodified probe and probes containingthe Q₂ or Q₁₈ nucleotide analogues. A higher signal to noise ratio wasachieved with the analogue probes, due to lower mismatch ligations. Thesignal to noise ratios with the Q₂ analogue appeared consistently higherthan the regular probes at the enzyme concentration up to 5 nM (100fmol). Furthermore, a higher signal to noise ratio was maintained atenzyme concentrations up to 10 nM (200 fmol) with the Q₁₈ analogue,indicating that Q₁₈ may be better than Q₂ in tolerating variations ofenzyme concentration.

Introduction of the Q₂ or Q₁₈ analogues at the 3rd position of thediscriminating probe improves the signal to noise ratio about 2 to3-fold, thereby increasing the power of the LDR system to discriminatecancer signal from background. This assay compares the ability of ligaseto discriminate the most difficult case; a T:G mismatch from an T:Aperfect match. The Q₂ or Q₁₈ analogues located three nucleotides in fromthe 3′-end of a probe enhance local melting when present in conjunctionwith a mismatch at the 3′-position, while at the same time preservinghelix integrity more than a mismatch when present in conjunction with abase pair match at the 3′-end. The use of a Q₂ or Q₁₈ analogue near the3′ end of a probe may influence the stability of the perfect matched andmismatched enzyme-DNA complexes to a different extent, so that discreteK_(M) effects are exerted on these ligation reactions. In a competitivereaction, such as ligation of perfectly matched and mismatchedsubstrates, the ratio of the specificity constant may be altered as aconsequence of K_(M), and possible k_(cat) changes for each substrate.All modified probes which satisfy the equation below (shown for Qanalogues) will give increased discrimination of cancer-associatedmutations in the presence of an excess of normal DNA.$\frac{\left\lbrack {k_{cat}/K_{M}} \right\rbrack_{{{SLP3}^{\prime}{QTT}},{match}}}{\left\lbrack {k_{cat}/K_{M}} \right\rbrack_{{{SLP3}^{\prime}{QTT}},{mismatch}}} > \frac{\left\lbrack {k_{cat}/K_{M}} \right\rbrack_{{{SLP3}^{\prime}{TTT}},{match}}}{\left\lbrack {k_{cat}/K_{M}} \right\rbrack_{{{SLP3}^{\prime}{TTT}},{mismatch}}}$

Alternatively, the third aspect of the present invention can beexpressed in terms of a fidelity ratio (i.e. the initial rate ofligating a substrate with an analogue located three nucleotides in fromthe 3′ end as well as a perfect match at the 3′ end divided by theinitial rate of ligating a substrate with an analogue located threenucleotides in from the 3′ end as well as a mismatch at the 3′ end) asfollows:${\frac{\left\lbrack k_{1} \right\rbrack_{{{SLP3}^{\prime}{QTT}},{match}}}{\left\lbrack k_{1} \right\rbrack_{{{SLP3}^{\prime}{QTT}},{mismatch}}} > \frac{\left\lbrack k_{1} \right\rbrack_{{{SLP3}^{\prime}{TTT}},{match}}}{\left\lbrack k_{1} \right\rbrack_{{{SLP3}^{\prime}{TTT}},{mismatch}}}} = {{Fidelity}\quad {ratio}}$

Detection of Target Containing a C:A Mismatch in the Presence of anExcess of Normal Template Using Wild Type and Mutant Tth DNA Ligases

The assay, which can quantify the amount of a low abundance sequence(cancer mutation) in an excess of normal DNA, was extended to determinethe limits of detecting a C:G match in the presence of an excess C:Amismatches. Using wild-type and mutant Tth DNA ligases in an LDR assay,the fraction of “cancer” DNA in a mixture of normal and cancer DNA wasquantified. The probes used to determine the limits of detection areshown in FIG. 26, with sequences in Table 7.

TABLE 7 Size (bp) Sequence (5′--->3′) Probe Name (Com610-3′F 30Fam-GGGTCTGATCTCCTAGTTTGATACTGTTGA SEQ. ID. No. 31) (SLP3′TTT 21ATGCGTCTGCGGTGTTGCTTT SEQ. ID. No. 30) (SLP3′TTC 23AAATGCGTCTGCGGTGTTGCTTC SEQ. ID. No. 29) Template Name (Glg.m3A 59CCCAGACTAGAGGATCAAACTATGACAACTGAA SEQ. ID. No 32)GCAACACCGCAGACGCTGGAACAGGG (Glg.m3A.Rev 59CCCTGTTCCAGCGTCTGCGGTGTTGCTTCAGTTGT SEQ. ID. No. 33)CATAGTTTGATCCTCTAGTCTGGG (Alg.m3A 59 CCCAGACTAGAGGATCAAACTATGACAACTAAASEQ. ID. No. 34) GCAACACCGCAGACGCTGGAACAGGG (Alg.m3A.Rev 59CCCTGTTCCAGCGTCTGCGGTGTTGCTTTAGTTGT SEQ. ID. No. 35)CATAGTTTGATCCTCTAGTCTGGG Table 7 shows LDR probes and Template sequencesfor a C:A mismatch in an excess of normal DNA by either wild-type ormutant K294R Tth DNA ligase. Probes ALg.m3A and ALg.m3A.rev representthe mismatched template (Normal template), whereas GLg.m3A andGLg.m3A.rev represent the perfect matched template (Cancer template).Probes SLP3′TTC represents Normal probe for the perfect matched (Cancer)template; whereas SLP3′TTT represents the Normal probe for themismatched (Normal) template.

Two oligonucleotides were hybridized to the target such that the 3′ endof the upstream probe (SLP3′TTC) is immediately adjacent to the 5′ endof the downstream probe (Com 610-3′F). The probes used in this reactioncreate a C:A mismatch on the “Normal” template and an C:G match on the“Cancer” template at the ligation junction. LDR experiments were done intriplicate using 42.5 nM (250 fmole) of the C:A mismatched template(“Normal”; ALg.m3A and Alg.m3Arev), containing from 0 to 2.5 nM (50fmole) of perfect matched template (“Cancer”; GLg.m3A and Glg.m3A.rev)in the presence of 25 fmol of purified wild-type and mutant/enzyme K294R(See FIG. 27). Both enzymes generated about the same amount of product,and the mutant K294R enzyme gave somewhat less background mismatchligation. Thus, the signal-to-noise ratio was only slightly improved forthe mutant enzyme compared to the wild-type ligase (See FIG. 28). Withthe K294R mutant enzyme, the signal-to-noise ratio was 2.2 fordistinguishing one “Cancer” template in 500 “Normal” template, andincreased to 3.1 for distinguishing one in 250 “Normal” templates (Seenumbers in FIG. 28, as well as a similar experiment described in Table8).

TABLE 8 Amount of Product Formed (fmol) with different Templates usingthe C/A mismatch 10 fmol C Template 2.5 fmol of C in 250 fmol of NTemplate in 250 fmol Tth Ligase Conc Template of N Template (fmol) 250fmol N Template 10 fmol C Template (1:25) (1:100) Mutant Enzyme 0.8233.26 14.53 6.62 K294 R (50 fmol) Wild Type Enzyme 1.48 35.69 16.70 7.60(50 fmol) Mutant Enzyme 0.95 36.52 17.91 5.66 K294 R (25 fmol) Wild TypeEnzyme 1.04 24.94 13.50 7.47 (25 fmol) Signal/Noise Ratio — — 17.72 8.07K294 R (50 fmol) Wild Type Enzyme — — 11.28 5.13 (50 fmol) Mutant Enzyme— — 18.85 5.95 K294 R (25 fmol) Wild Type Enzyme — — 12.98 7.19 (25fmol) Table 8. Comparison of the wild type and mutant Tth ligase indetecting the Cancer signal in the presence of 25 and 100 fold excess ofthe normal template (C:A mismatch). N = Normal template; ALg.m3A andAlg.m3A rev. C = Cancer template; GLg.m3A and GLg.m3A rev; The fourdifferent conditions used in a ligation detection reaction was asfollows (i) 12.5 nM (250 fmol) of Normal (mismatch) Template (ii) 0.5 nM(10 fmol) of Cancer (perfect match) Template (iii) 0.5 nM (10 fmol) of #Cancer Template in 12.5 nM (250 fmol) of Normal Template and (iv) 0.125nM (2.5 fmol) of Cancer Template in 12.5 nM (250 fmol) of NormalTemplate. The primers used in this reaction create a C:A mismatch on the“Normal” template and an C:G match on the “Cancer” template at theligation junction. Each reaction was carried out in a 20 μl volume of areaction buffer containing 25 nM (500 fmol) of each of the two regularprimrs (SLP3′TTC and Com 610 3′F), using 2.5 nM # (50 fmol); and 1.25 nM(25 fmol), of either the wild-type or the mutant enzyme. Data wasanalyzed using the Genescan 672 software. In calculating the signal tonoise ratios, the signal was defined as the amount of perfect matchproduct formed in the presence of 25- or 100-fold N template, the valueof noise was taken as the amount of mismatch product formed with 12.5 nM(250 fmol) N template.

Can conditions where the mutant K294R enzyme is superior to wild-typeenzyme in distinguishing a C:A mismatch be found? Initial experiments onLDR were done in triplicate using the mismatched (“Normal”) template andperfect match (“Cancer”) template independently, or in combination witheach other in the presence of 25 and 50 fmol of purified wild-type andmutant enzyme K294R (Table 8). The results obtained with the C:Amismatch were comparable to the results obtained with the T:G mismatchusing 25 fmol of enzyme. However, when using the higher enzyme amount(50 fmoles), the amount of LDR product generated by the wild-type enzymeon the mismatch template was at least 1.8 fold greater than generated bythe mutant enzyme. LDR was also performed with the perfect match andmismatch templates together in a ratio of 1:25 and 1:100, respectively.For both wild type and mutant enzymes product generated in the presenceof the mismatch template was less than the product generated by theperfect match template alone. The mutant K294R, generated slightly lessproduct than the wild type enzyme under identical conditions, however,combined with a lower background signal from the normal template, theuse of K294R results in about a 1.5 to 2-fold higher signal-to-noiseratio. Hence these results are in conjunction with our previous resultsand support the earlier finding that the mutant K294R exhibits a higherfidelity in discriminating a mismatch over the wild type enzyme. We alsonote that 50 fmoles of enzyme gave the best signal-to-noise ratio whenusing Q analogue containing probes (Table 8).

Example 18

Quantitative Detection of Target Containing a Single-Base Mutation, inthe Presence of an Excess of Normal Template, Using Wild-Type and MutantTth Ligases

An assay has been developed to quantify the amount of cancer mutation ina given sample. Using wild-type and mutant Tth ligases in an LDR assay,the fraction of “cancer” DNA in a mixture of normal and cancer DNA wasquantified. The probes used to determine the limits of detection areshown in FIG. 30. Two oligonucleotides were hybridized to the targetsuch that the 3′ end of the upstream probe is immediately adjacent tothe 5′ end of downstream probe. Tth DNA ligase can then join the twoadjacent oligonucleotides, provided that the nucleotides at the junctionare correctly base-paired with the target strand. LDR experiments weredone in triplicate using 12.5 nM (250 fmole) of the mismatched template(“Normal”), containing from 0 to 2.5 nM (50 fmole) of perfect matchedtemplate (“Cancer”) in the presence of 25 fmol of purified wild-type andmutant enzyme K294R (See FIG. 21). Both enzymes generated about the sameamount of product, but the mutant K294R enzyme gave less backgroundmismatch ligation. Therefore, the signal-to-noise ratio was somewhatbetter (about 1.5-fold) for the mutant enzyme compared to the wild-typeligase (FIG. 22). With the K294R mutant enzyme, the signal-to-noiseratio was 3.3 for distinguishing one “Cancer” template in 500 “Normal”templates and increased to 5.4 for distinguishing one in 250 “Normal”templates. This assay compares the ability of ligase to discriminate themost difficult case; a G:T mismatch from an A:T perfect match. Since theTth DNA ligase shows five-fold greater fidelity in distinguishing a C:Amismatch (the second most difficult case) compared to a G:T mismatch,the mutant enzyme would be predicted to distinguish all other mismatchesat 1 in 2,500 or better.

The experiment using synthetic substrates which generate a C:A mismatchhas been carried out (See Table 7), and the limits of detection andsignal to noise values were determined (see FIGS. 27 and 29). Controlligations to quantify the amount of normal DNA gave qualitativelysimilar results as before (see FIG. 28). There was no significantdifference between wild-type and mutant enzyme with these substrates.With the K294R mutant enzyme, the signal-to-noise ratio was 2.2 fordistinguishing one “Cancer” template in 500 “Normal” templates, andincreased to 3.1 for distinguishing one in 250 “Normal” templates.Although the synthetic substrate results suggest there is no fundamentaldifference in distinguishing a G:T mismatch from a C:A mismatch,experiments with natural PCR products containing the K-ras gene suggestthat significantly better discrimination may be achieved with C:Amismatches than with G:T mismatches.

Example 19

Design and Synthesis of LDR Probes to Detect all Possible Mutations inCodons 12, 13, and 61 of the K-ras Oncogene

The K-ras gene presents two significant challenges for mutationdetection techniques. Extensive sequence homology between the H-, N- andK-ras genes requires a primary exon-specific PCR reaction to amplifyselectively the correct gene. A subsequent allele-specific PCR has beenused to detect individual K-ras mutations, but a multiplex PCR to detectall mutations is complicated by the proximity of codon 12 and codon 13mutations.

The scheme for simultaneously assaying mutations in codons 12, 13, and61 of the K-ras gene is shown in FIG. 30. Two independent PCR primerpairs which correctly PCR amplify the K-ras gene in the regionssurrounding codons 12 and 13, and codon 61 have been synthesized. Allpossible LDR probes for these codons were designed and synthesized(FIGS. 31A-B). The discriminating oligonucleotides have calculated T_(m)values of about 66° C. and contain the discriminating bases at their 3′ends. These oligonucleotides generate products distinguished byalternating FAM- or TET-fluorescent peaks when separated on an automatedABI 373A DNA sequencer. The common oligonucleotides have calculatedT_(m) values of about 69° C. and contain poly-A tails with spacer C3blocking groups at their 3′ ends. The blocking groups prevent residualpolymerase extension of LDR primers, and the poly-A tails allow the LDRproducts to be separated on sequencing gels. Common probes werechemically phosphorylated at their 5′ ends.

The LDR probes were designed such that the most common mutations wouldgenerally provide the smaller ligation products. Thus, for the sevenmost common K-ras mutations in colon cancer, the products would be 44 bpfor G12D (GAT), 45 bp for G12A (GCT), 46 bp for G12V (GTT), 47 bp forG12S (AGT), 48 bp for G12R (CGT), 49 bp for G12C (TGT), and 51 bp forG13D (GAC). Probes specific for wild-type DNA have also been designed toquantify the amount of PCR product prior to detection of mutant DNA. PCRproduct is estimated by ethidium bromide staining in a 3% agarose gel.

Example 20

Testing PCR/LDR to Identify K-ras Codons 12, 13, and 61 Mutations in aMultiplex Format

This experiment verifies that all LDR probes can be multiplexed fordetecting the mutations at codons 12, 13, and 61 of K-ras. DNA wasprepared from cell lines with known mutations in codons 12 and 13(SW620, G12V; SW1116, G12A; LS180, G12D; DLD1, G13D). PCR/LDR usingindividual probes complementary to the particular mutation yielded thecorrect ligation products. Furthermore, equal mixing of two DNA targetscontaining separate mutations (G12D and G12V) in an excess of normal DNAgave both LDR products in roughly equimolar amounts.

The sensitivity of PCR/LDR was determined by reconstructing samplescontaining various dilutions of mutant DNA derived from cell lines inwild-type DNA. Samples were PCR amplified independently, and then mixed,allowing the testing of a variety of conditions. Exploratory experimentsallowed optimization of the total amount of normal and mutant DNA (2,000fmoles total in 20 μl reaction), fluorescently labeled discriminatingprobes (500 fmoles each), common probes (from 500 to 1,500 fmoles each),and finally Tth DNA ligase (100 fmoles of either wild-type or K294Rmutant enzyme). A standard PCR/LDR reaction included 30 PCR cycles and20 LDR cycles.

In an initial experiment, from 0.025 nM (0.5 fmol) to 5 nM (100 fmol) ofPCR amplified DNA containing the G12D mutation was diluted into 100 nM(2000 fmol) of wild-type PCR amplified DNA. The amount of LDR productformed with either wild-type or K294R mutant enzyme was approximatelythe same, with similar signal-to-noise values (See FIGS. 33 and 34).With the K294R mutant enzyme, the signal-to-noise ratio was 1.7 fordistinguishing one G12D template in 2,000 wild-type templates, andincreased to 3.2 for distinguishing one in 1,000 wild-type templates.

When probe sets are combined to test all six possible single-basemutations at K-ras codon 12 in a multiplexed LDR reaction, the greatestbackground noise was obtained from probes designed to detect G12D, i.e.representing a C:A mismatch. Therefore, for determining signal-to-noisevalues in a multiplexed assay, the G12V mutant was used so that both“signal” (from the G12V) and “noise” (C:A misligations from the G12Dprobes on wild-type template) could be quantified in the same reaction.For the six probe set, similar amounts of product were formed with bothenzymes, with the K294 mutant demonstrating slightly bettersignal-to-noise values (See FIGS. 35-38). However, with 20 fmol, 40fmol, 80 fmol, 100 fmol, and 200 fmol of cancer template in 2000 fmol ofwild-type DNA, the signal to noise ratio obtained w/K294R weresignificantly higher than obtained with wild type ligase. With the K294Rmutant enzyme, the signal-to-noise ratio was 3.2 for distinguishing oneG12V template in 1,000 wild-type templates and increased to 5.5 fordistinguishing one in 500 wild-type templates. Thus, surprisingly,despite the potential for interference during hybridization, thesignal-to-noise values obtained with the six mutation multiplex reactionare comparable to those obtained with a single probe set at the lowestdilutions. At higher cancer DNA concentrations, the interference betweenoverlapping probes becomes more apparent as less signal is generatedwhen using the full complement of all 26 probes compared to using either8 probes or a single set of probes.

The full complement of all 26 probes was used to test all 19 possiblesingle-base mutations at K-ras codons 12, 13 and 61 in a multiplexed LDRreaction. The background noise for probes designed to detect Q₆₁R, i.e.representing a G:T mismatch was about 10-fold higher than that observedfor probes designed to detect G12D, i.e. representing a C:A mismatch.Probes may be designed to avoid G:T mismatches by using the oppositestrand sequences. For the 26 probe set, similar amounts of product wereformed with both enzymes, although they were clearly less than with the6 probe set (See FIGS. 39-40). With the K294R mutant enzyme, thesignal-to-noise ratio (comparing to the G12D C:A misligation) was 3.2for distinguishing one G12V template in 500 wild-type templates, andincreased to 7.2 for distinguishing one in 250 wild-type templates.Thus, use of 16 overlapping probes which hybridize to codons 12 and 13does reduce both overall signal, as well as signal to noise, yet canstill distinguish one mutation in 500. While others have reported usingT4 ligase to detect 3 mutations in codon 12 with a 1% sensitivity(requiring blocking oligonucleotides and high salt to suppressmis-ligations on wild-type template), thermostable enzyme gives fargreater sensitivity for simultaneously detecting all 12 mutations incodons 12 and 13. Powell et. al., N.E. J. Med. 329:1982-87 (1993), Jen,et. al., Cancer Res. 54:5523-26 (1994), and Redston, et. al., Gastroent.108:383-92 (1995), which are hereby incorporated by reference.

Use has been made of PCR-amplified templates from microdissected tissue,for which the K-ras mutations have already been determined using directsequencing. In blinded experiments, 148 coded samples, mostly containingK-ras mutations were provided to determine which mutation is presentusing PCR/LDR. Presence of a single mutation was clearly indicated byappearance of a single band migrating at a given length (See FIGS.41-42). By comparing observed length with expected LDR product length,the mutation was determined (Table 9, and FIG. 40).

TABLE 9 LDR Product K-ras Mutation 44-Fam Asp-12 45-Tet Ala-12 46-FamVal-12 47-Tet Ser-12 48-Fam Arg-12 49-Tet Cys-12 51-Fam Asp-13 52-TetAla-13 53-Fam Val-13 54-Tet Ser-13 55-Fam Arg-13 56-Tet Cys-13 59-TetHis-61 60-Fam His-61 61-Tet Arg-61 62-Fam Leu-61 63-Tet Pro-61 64-FamLys-61 65-Tet Glu-61 Table 9 lists the expected LDR products which wouldbe produced in an LDR reaction containing 25 nM (500 fmol) of 19discriminating probes (Tet-K-ras c12.2A, Tet-K-ras c12.1S, Tet-K-rasc12.1C, Tet-K-ras c13.4A, Tet-K-ras c13.3S, Tet-K-ras c13.3C, Tet-K-rasc61.7HT, Tet-K-ras c61.6R, Tet-K-ras c61.5K, Tet-K-ras c61.6P, Fam-K-rasc12.1R, Fam-K-ras c12.2D, Fam-K-ras c12.2V, Fam-K-ras c13.4D, Fam-K-rasc13.4V, Fam-K-ras c13.3R, Fam-K-ras c61.7HC, Fam-K-ras c61.6L, Fam-K-rasc61.5K); 50 nM # (1000 fmol) of two common probes (K-ras c61 Com-7 andK-ras c12 Com-5); and 75 nM (1500 fmol) of five common probes (K-ras c12Com-2, K-ras c12 Com-1, K-ras c13 Com-4, K-ras c13 Com-3, and K-ras c61Com-6) and 5 nM (100 fmol) of the wild-type or K294R mutant enzymes. Thelength and label of the LDR product listed in the left columncorresponds to the presence in the sample of the specific K-ras mutationlisted in the right column.

Of the 148 unknown samples and 10 known samples tested, there wasagreement on all known samples and 138 unknown samples. FIG. 43 is atable comparing the 10 discordant samples. The results obtained bymultiplex PCR/LDR were confirmed by PCR/LDR using only the LDR probe setspecific to the mutation. Nine of the ten discordant samples wereaccurately typed by PCR/LDR which only missed a single, very rare doublemutation found by sequencing. In other experiments, detection ofmutations was compared in micro-dissected tumors versus whole paraffinsections containing both normal and tumor tissue. Multiplex PCR/LDRdetected all known mutations even without the requirement formicro-dissection.

Thus, the utility of multiplexed PCR/LDR on samples derived from celllines, microdissected tumors, and paraffin embedded tissues has beenshown. The technique is both highly accurate (99.3%) and sensitive (i.e.able to detect 1 mutation in 500 normal sequences).

Example 21

Use of Low Level Normal Probes to Provide Low Level LDR Products WhichQuantify Total Normal DNA in Sample

LDR reactions were run for 15 sec at 94° C., and 4 min. at 65° C. percycle for 20 cycles. The reactions were completely stopped by chillingthe tubes in an ethanol-dry ice bath and adding 0.5 μl of 0.5 mM EDTA.Aliquots of 2.5 μl of the reaction products were mixed with 2.5 μl ofloading buffer (83% Formamide, 8.3 mM EDTA, and 0.17% Blue Dextran) and0.5 μl GeneScan TAMRA 350 molecular weight marker, denatured at 94° C.for 2 min, chilled rapidly on ice prior to loading on an 8 M urea-10%polyacrylamide gel, and electrophoresed on an ABI 373 DNA sequencer at1400 volts. Fluorescent ligation products were analyzed and parametersof an exponential equation fit to the data using the Deltagraph Pro 3.5.software.

FIGS. 44-45 show the quantitative detection of different amounts ofK-ras Normal template when varying amounts of wild-type probes were usedby either wild-type or K294R Tth DNA ligase. The amount of LDR productformed when 25 nM (500 fmol), 50 nM (1000 fmol), and 100 nM (2000 fmol)of the “Normal” template was reacted with 0.5 nM (10 fmol), 2.5 nM (50fmol), and 5 nM (100 fmol) of the wild type discriminating probe(Tet-K-ras c12.2 WtG) and common probe (K-ras c12 Com-2) in the presenceof 25 nM (500 fmol) of nineteen discriminating probes (Tet-K-ras c12.2A,Tet-K-ras c12.1S, Tet-K-ras c12.1C, Tet-K-ras c13.4A, Tet-K-ras c13.3S,Tet-K-ras c13.3C, Tet-K-ras c61.7HT, Tet-K-ras c61.6R, Tet-K-ras c61.5K,Tet-K-ras c61.6P, Fam-K-ras c12.1R, Fam-K-ras c12.2D, Fam-K-ras c 12.2V,Fam-K-ras c13.4D, Fam-K-ras c13.4V, Fam-K-ras c13.3R, Fam-K-ras c61.7HC,Fam-K-ras c61.6L, Fam-K-ras c61.5K); 50 nm (1000 fmol) of two commonprobes (K-ras c61 Com-7 and K-ras c12 Com-5); and 75 nm (1500 fmol) offive common probes (K-ras c12 Com-2, K-ras c12 Com-1, K-ras c13 Com4,K-ras c13 Com-3, and K-ras c61 Com-6) and 5 nm (100 fmol) of the wildtype or K294R mutant enzymes. The X-axis indicated the different amountsof the Normal Template, while the Y-axis indicated the amount of LDRproduct generated. (▪, Δ, □) represents 0.5 nM (10 fmol), 2.5 nM (50fmol), and 5 nM (100 fmol), respectively, of the wild type probes usedwith the wild-type enzyme whereas (, ♦, ∘) represents 0.5 (10 fmol),2.5 nM (50 fmol), and 5 nM (100 fmol) of the wild type probes used withthe K294R mutant enzyme.

Although the invention has been described in detail for the purpose ofillustration, it is understood that such details are solely for thatpurpose and that variations can be made therein by those skilled in theart without departing from the spirit of the scope of the inventionwhich is defined by the following claims.

97 1 18 DNA Artificial Sequence Description of Artificial SequencePrimer for PCR or LDR 1 gaccctggaa gaggcgag 18 2 27 DNA ArtificialSequence Description of Artificial Sequence Primer for PCR or LDR 2cgtccacsng gtgctccacg gtgtagg 27 3 28 DNA Artificial SequenceDescription of Artificial Sequence Primer for PCR or LDR 3 tggagcaccnsgtggacggg ctttccgt 28 4 17 DNA Artificial Sequence Description ofArtificial Sequence Primer for PCR or LDR 4 gcaaactggg tcgccac 17 5 29DNA Artificial Sequence Description of Artificial Sequence Primer forPCR or LDR 5 gaaagcccgt dcaccttgtg ctccacggt 29 6 26 DNA ArtificialSequence Description of Artificial Sequence Primer for PCR or LDR 6acaaggtgha cgggctttcc gtgaac 26 7 29 DNA Artificial Sequence Descriptionof Artificial Sequence Primer for PCR or LDR 7 gaaagccctn ccaccttgtgctccacggt 29 8 28 DNA Artificial Sequence Description of ArtificialSequence Primer for PCR or LDR 8 acaaggtggn agggctttcc gtgaacct 28 9 28DNA Artificial Sequence Description of Artificial Sequence Primer forPCR or LDR 9 ccctgttcca gcgtctgcgg tgttgcgt 28 10 37 DNA ArtificialSequence Description of Artificial Sequence Primer for PCR or LDR 10aagttgtcat agtttgatcc tctagtctgg gaaaaaa 37 11 35 DNA ArtificialSequence Description of Artificial Sequence Primer for PCR or LDR 11cagttgtcat agtttgatcc tctagtctgg gaaaa 35 12 33 DNA Artificial SequenceDescription of Artificial Sequence Primer for PCR or LDR 12 gagttgtcatagtttgatcc tctagtctgg gaa 33 13 31 DNA Artificial Sequence Descriptionof Artificial Sequence Primer for PCR or LDR 13 tagttgtcat agtttgatcctctagtctgg g 31 14 59 DNA Artificial Sequence Description of ArtificialSequence Primer for PCR or LDR 14 gggacaaggt cgcagacgcc acaacgcaatcaacagtatc aaactaggag atcagaccc 59 15 30 DNA Artificial SequenceDescription of Artificial Sequence Primer for PCR or LDR 15 agttgtcatagtttgatcct ctagtctggg 30 16 35 DNA Artificial Sequence Description ofArtificial Sequence Primer for PCR or LDR 16 aaaaaaccct gttccagcgtctgcggtgtt gcgta 35 17 33 DNA Artificial Sequence Description ofArtificial Sequence Primer for PCR or LDR 17 aaaaccctgt tccagcgtctgcggtgttgc gtc 33 18 31 DNA Artificial Sequence Description ofArtificial Sequence Primer for PCR or LDR 18 aaccctgttc cagcgtctgcggtgttgcgt g 31 19 29 DNA Artificial Sequence Description of ArtificialSequence Primer for PCR or LDR 19 ccctgttcca gcgtctgcgg tgttgcgtt 29 2059 DNA Artificial Sequence Description of Artificial Sequence Primer forPCR or LDR 20 gggacaaggt cgcagacgcc acaacgcagt caacagtatc aaactaggagatcagaccc 59 21 59 DNA Artificial Sequence Description of ArtificialSequence Primer for PCR or LDR 21 gggacaaggt cgcagacgcc acaacgcattcaacagtatc aaactaggag atcagaccc 59 22 59 DNA Artificial SequenceDescription of Artificial Sequence Primer for PCR or LDR 22 gggacaaggtcgcagacgcc acaacgcact caacagtatc aaactaggag atcagaccc 59 23 20 DNAArtificial Sequence Description of Artificial Sequence Primer for PCR orLDR 23 tacgtctgcg gtgttgcgtc 20 24 18 DNA Artificial SequenceDescription of Artificial Sequence Primer for PCR or LDR 24 cgtctgcggtgttgcgtt 18 25 21 DNA Artificial Sequence Description of ArtificialSequence Primer for PCR or LDR 25 atgcgtctgc ggtgttgcat c 21 26 19 DNAArtificial Sequence Description of Artificial Sequence Primer for PCR orLDR 26 gcgtctgcgg tgttgcatt 19 27 23 DNA Artificial Sequence Descriptionof Artificial Sequence Primer for PCR or LDR 27 aaatgcgtct gcggtgttgcntc 23 28 21 DNA Artificial Sequence Description of Artificial SequencePrimer for PCR or LDR 28 atgcgtctgc ggtgttgcnt t 21 29 23 DNA ArtificialSequence Description of Artificial Sequence Primer for PCR or LDR 29aaatgcgtct gcggtgttgc ttc 23 30 21 DNA Artificial Sequence Descriptionof Artificial Sequence Primer for PCR or LDR 30 atgcgtctgc ggtgttgctt t21 31 30 DNA Artificial Sequence Description of Artificial SequencePrimer for PCR or LDR 31 agttgtcata gtttgatcct ctagtctggg 30 32 59 DNAArtificial Sequence Description of Artificial Sequence Primer for PCR orLDR 32 gggacaaggt cgcagacgcc acaacgaagt caacagtatc aaactaggag atcagaccc59 33 59 DNA Artificial Sequence Description of Artificial SequencePrimer for PCR or LDR 33 ccctgttcca gcgtctgcgg tgttgcttca gttgtcatagtttgatcctc tagtctggg 59 34 59 DNA Artificial Sequence Description ofArtificial Sequence Primer for PCR or LDR 34 gggacaaggt cgcagacgccacaacgaaat caacagtatc aaactaggag atcagaccc 59 35 59 DNA ArtificialSequence Description of Artificial Sequence Primer for PCR or LDR 35ccctgttcca gcgtctgcgg tgttgcttta gttgtcatag tttgatcctc tagtctggg 59 3659 DNA Artificial Sequence Description of Artificial Sequence Primer forPCR or LDR 36 gggacaaggt cgcagacgcc acaacgcagt caacagtatc aaactaggagatcagaccc 59 37 59 DNA Artificial Sequence Description of ArtificialSequence Primer for PCR or LDR 37 ccctgttcca gcgtctgcgg tgttgcgtcagttgtcatag tttgatcctc tagtctggg 59 38 20 DNA Artificial SequenceDescription of Artificial Sequence Primer for PCR or LDR 38 tacgtctgcggtgttgcgtc 20 39 18 DNA Artificial Sequence Description of ArtificialSequence Primer for PCR or LDR 39 cgtctgcggt gttgcgtt 18 40 59 DNAArtificial Sequence Description of Artificial Sequence Primer for PCR orLDR 40 gggacaaggt cgcagacgcc acaacgtagt caacagtatc aaactaggag atcagaccc59 41 59 DNA Artificial Sequence Description of Artificial SequencePrimer for PCR or LDR 41 ccctgttcca gcgtctgcgg tgttgcatca gttgtcatagtttgatcctc tagtctggg 59 42 21 DNA Artificial Sequence Description ofArtificial Sequence Primer for PCR or LDR 42 atgcgtctgc ggtgttgcnt t 2143 21 DNA Artificial Sequence Description of Artificial Sequence Primerfor PCR or LDR 43 atgcgtctgc ggtgttgcnt t 21 44 21 DNA ArtificialSequence Description of Artificial Sequence Primer for PCR or LDR 44atgcgtctgc ggtgttgcnt t 21 45 20 DNA Artificial Sequence Description ofArtificial Sequence Primer for PCR or LDR 45 atgcgtctgc ggtgttgcnc 20 4621 DNA Artificial Sequence Description of Artificial Sequence Primer forPCR or LDR 46 atgcgtctgc ggtgtnnngt t 21 47 21 DNA Artificial SequenceDescription of Artificial Sequence Primer for PCR or LDR 47 atgcgtctgcggtgtnnngt c 21 48 20 DNA Artificial Sequence Description of ArtificialSequence Primer for PCR or LDR 48 acgcagacgc cacaacgcaa 20 49 24 DNAArtificial Sequence Description of Artificial Sequence Primer for PCR orLDR 49 aaaacttgtg gtagttggag ctga 24 50 25 DNA Artificial SequenceDescription of Artificial Sequence Primer for PCR or LDR 50 caaaacttgtggtagttgga gctgc 25 51 27 DNA Artificial Sequence Description ofArtificial Sequence Primer for PCR or LDR 51 acaaaaactt gtggtagttggagctgt 27 52 20 DNA Artificial Sequence Description of ArtificialSequence Primer for PCR or LDR 52 tggcgtaggc aagagtgcct 20 53 26 DNAArtificial Sequence Description of Artificial Sequence Primer for PCR orLDR 53 atataaactt gtggtagttg gagcta 26 54 27 DNA Artificial SequenceDescription of Artificial Sequence Primer for PCR or LDR 54 aatataaacttgtggtagtt ggagctc 27 55 28 DNA Artificial Sequence Description ofArtificial Sequence Primer for PCR or LDR 55 caatataaac ttgtggtagttggagctt 28 56 22 DNA Artificial Sequence Description of ArtificialSequence Primer for PCR or LDR 56 gtggcgtagg caagagtgcc aa 22 57 21 DNAArtificial Sequence Description of Artificial Sequence Primer for PCR orLDR 57 tgtggtagtt ggagctggtg a 21 58 22 DNA Artificial SequenceDescription of Artificial Sequence Primer for PCR or LDR 58 atgtggtagttggagctggt gc 22 59 23 DNA Artificial Sequence Description of ArtificialSequence Primer for PCR or LDR 59 aatgtggtag ttggagctgg tgt 23 60 30 DNAArtificial Sequence Description of Artificial Sequence Primer for PCR orLDR 60 cgtaggcaag agtgccttga caaaaaaaaa 30 61 22 DNA Artificial SequenceDescription of Artificial Sequence Primer for PCR or LDR 61 cttgtggtagttggagctgg ta 22 62 22 DNA Artificial Sequence Description of ArtificialSequence Primer for PCR or LDR 62 acttgtggta gttggagctg gt 22 63 24 DNAArtificial Sequence Description of Artificial Sequence Primer for PCR orLDR 63 aacttgtggt agttggagct ggtt 24 64 32 DNA Artificial SequenceDescription of Artificial Sequence Primer for PCR or LDR 64 gcgtaggcaagagtgccttg aaaaaaaaaa aa 32 65 25 DNA Artificial Sequence Description ofArtificial Sequence Primer for PCR or LDR 65 agatattctc gacacagcag gtcat25 66 26 DNA Artificial Sequence Description of Artificial SequencePrimer for PCR or LDR 66 aagatattct cgacacagca ggtcac 26 67 34 DNAArtificial Sequence Description of Artificial Sequence Primer for PCR orLDR 67 gaggagtaca gtgcaatgag ggacaaaaaa aaaa 34 68 23 DNA ArtificialSequence Description of Artificial Sequence Primer for PCR or LDR 68gatattctcg acacagcagg tcg 23 69 24 DNA Artificial Sequence Descriptionof Artificial Sequence Primer for PCR or LDR 69 agatattctc gacacagcaggtct 24 70 25 DNA Artificial Sequence Description of Artificial SequencePrimer for PCR or LDR 70 aagatattct cgacacagca ggtcc 25 71 38 DNAArtificial Sequence Description of Artificial Sequence Primer for PCR orLDR 71 agaggagtac agtgcaatga gggaaaaaaa aaaaaaaa 38 72 23 DNA ArtificialSequence Description of Artificial Sequence Primer for PCR or LDR 72ggatattctc gacacagcag gta 23 73 24 DNA Artificial Sequence Descriptionof Artificial Sequence Primer for PCR or LDR 73 aggatattct cgacacagcaggtg 24 74 41 DNA Artificial Sequence Description of Artificial SequencePrimer for PCR or LDR 74 aagaggagta cagtgcaatg agggcaaaaa aaaaaaaaaa a41 75 31 DNA Artificial Sequence Description of Artificial SequencePrimer for PCR or LDR 75 aaccacaggc tgctgcggat gccggtcgga g 31 76 30 DNAArtificial Sequence Description of Artificial Sequence Primer for PCR orLDR 76 agagccgcca ccctcagaac cgccaccctc 30 77 61 DNA Artificial SequenceDescription of Artificial Sequence Primer for PCR or LDR 77 gagggtggcggttctgaggg tggcggctct ctccgaccgg catccgcagc agcctgtggt 60 t 61 78 18 DNAArtificial Sequence Description of Artificial Sequence primer for PCR orLDR 78 cagaacctcc tcaccatc 18 79 27 DNA Artificial Sequence Descriptionof Artificial Sequence primer for PCR or LDR 79 ctcgtccagn ngcaccaccaccccgtc 27 80 28 DNA Artificial Sequence Description of ArtificialSequence primer for PCR or LDR 80 cgcccggttt nccccacctg gaagacca 28 8126 DNA Artificial Sequence Description of Artificial Sequence primer forPCR or LDR 81 gtcacccggn cggtgcgccc cacctg 26 82 27 DNA ArtificialSequence Description of Artificial Sequence primer for PCR or LDR 82ctcgtccagc ttcaccacca ccccgtc 27 83 24 DNA Artificial SequenceDescription of Artificial Sequence primer for PCR or LDR 83 tcgggcnnggtctcgggcca gcga 24 84 24 DNA Artificial Sequence Description ofArtificial Sequence primer for PCR or LDR 84 gtggcccnnc tcggggcagg tctc24 85 26 DNA Artificial Sequence Description of Artificial Sequenceprimer for PCR or LDR 85 gttgggcnng cggtggacct tcccct 26 86 25 DNAArtificial Sequence Description of Artificial Sequence primer for PCR orLDR 86 ggcgggcnnc aaggggttgg ggcag 25 87 26 DNA Artificial SequenceDescription of Artificial Sequence primer for PCR or LDR 87 acaaggtgnacgggctttcc gtgaac 26 88 28 DNA Artificial Sequence Description ofArtificial Sequence primer for PCR or LDR 88 tggtggtgcn nctggacgagcttgccct 28 89 25 DNA Artificial Sequence Description of ArtificialSequence primer for PCR or LDR 89 ggtggggnaa accgggcgtg tgacc 25 90 24DNA Artificial Sequence Description of Artificial Sequence primer forPCR or LDR 90 cgcaccgncc gggtgacccc tgtg 24 91 28 DNA ArtificialSequence Description of Artificial Sequence primer for PCR or LDR 91tggtggtgaa gctggacgag cttgccct 28 92 26 DNA Artificial SequenceDescription of Artificial Sequence primer for PCR or LDR 92 cccgagaccnngcccgagtg cggcca 26 93 28 DNA Artificial Sequence Description ofArtificial Sequence primer for PCR or LDR 93 tgccccgagn ngggccaccgcctcctca 28 94 28 DNA Artificial Sequence Description of ArtificialSequence primer for PCR or LDR 94 gtccaccgcn ngcccaaccc cttgtgcc 28 9528 DNA Artificial Sequence Description of Artificial Sequence primer forPCR or LDR 95 aaccccttgn ngcccgccaa gcgctttg 28 96 21 DNA ArtificialSequence Description of Artificial Sequence primer for PCR or LDR 96ctctatgtag ctctcgttgt g 21 97 676 PRT Thermus thermophilus 97 Met ThrLeu Glu Glu Ala Arg Lys Arg Val Asn Glu Leu Arg Asp Leu 1 5 10 15 IleArg Tyr His Asn Tyr Arg Tyr Tyr Val Leu Ala Asp Pro Glu Ile 20 25 30 SerAsp Ala Glu Tyr Asp Arg Leu Leu Arg Glu Leu Lys Glu Leu Glu 35 40 45 GluArg Phe Pro Glu Leu Lys Ser Pro Asp Ser Pro Thr Leu Gln Val 50 55 60 GlyAla Arg Pro Leu Glu Ala Thr Phe Arg Pro Val Arg His Pro Thr 65 70 75 80Arg Met Tyr Ser Leu Asp Asn Ala Phe Asn Leu Asp Glu Leu Lys Ala 85 90 95Phe Glu Glu Arg Ile Glu Arg Ala Leu Gly Arg Lys Gly Pro Phe Ala 100 105110 Tyr Thr Val Glu His Lys Val Asp Gly Leu Ser Val Asn Leu Tyr Tyr 115120 125 Glu Glu Gly Val Leu Val Tyr Gly Ala Thr Arg Gly Asp Gly Glu Val130 135 140 Gly Glu Glu Val Thr Gln Asn Leu Leu Thr Ile Pro Thr Ile ProArg 145 150 155 160 Arg Leu Lys Gly Val Pro Glu Arg Leu Glu Val Arg GlyGlu Val Tyr 165 170 175 Met Pro Ile Glu Ala Phe Leu Arg Leu Asn Glu GluLeu Glu Glu Arg 180 185 190 Gly Glu Arg Ile Phe Lys Asn Pro Arg Asn AlaAla Ala Gly Ser Leu 195 200 205 Arg Gln Lys Asp Pro Arg Ile Thr Ala LysArg Gly Leu Arg Ala Thr 210 215 220 Phe Tyr Ala Leu Gly Leu Gly Leu GluGlu Val Glu Arg Glu Gly Val 225 230 235 240 Ala Thr Gln Phe Ala Leu LeuHis Trp Leu Lys Glu Lys Gly Phe Pro 245 250 255 Val Glu His Gly Tyr AlaArg Ala Val Gly Ala Glu Gly Val Glu Ala 260 265 270 Val Tyr Gln Asp TrpLeu Lys Lys Arg Arg Ala Leu Pro Phe Glu Ala 275 280 285 Asp Gly Val ValVal Lys Leu Asp Glu Leu Ala Leu Trp Arg Glu Leu 290 295 300 Gly Tyr ThrAla Arg Ala Pro Arg Phe Ala Ile Ala Tyr Lys Phe Pro 305 310 315 320 AlaGlu Glu Lys Glu Thr Arg Leu Leu Asp Val Val Phe Gln Val Gly 325 330 335Arg Thr Gly Arg Val Thr Pro Val Gly Ile Leu Glu Pro Val Phe Leu 340 345350 Glu Gly Ser Glu Val Ser Arg Val Thr Leu His Asn Glu Ser Tyr Ile 355360 365 Glu Glu Leu Asp Ile Arg Ile Gly Asp Trp Val Leu Val His Lys Ala370 375 380 Gly Gly Val Ile Pro Glu Val Leu Arg Val Leu Lys Glu Arg ArgThr 385 390 395 400 Gly Glu Glu Arg Pro Ile Arg Trp Pro Glu Thr Cys ProGlu Cys Gly 405 410 415 His Arg Leu Leu Lys Glu Gly Lys Val His Arg CysPro Asn Pro Leu 420 425 430 Cys Pro Ala Lys Arg Phe Glu Ala Ile Arg HisPhe Ala Ser Arg Lys 435 440 445 Ala Met Asp Ile Gln Gly Leu Gly Glu LysLeu Ile Glu Arg Leu Leu 450 455 460 Glu Lys Gly Leu Val Lys Asp Val AlaAsp Leu Tyr Arg Leu Arg Lys 465 470 475 480 Glu Asp Leu Val Gly Leu GluArg Met Gly Glu Lys Ser Ala Gln Asn 485 490 495 Leu Leu Arg Gln Ile GluGlu Ser Lys Lys Arg Gly Leu Glu Arg Leu 500 505 510 Leu Tyr Ala Leu GlyLeu Pro Gly Val Gly Glu Val Leu Ala Arg Asn 515 520 525 Leu Ala Ala ArgPhe Gly Asn Met Asp Arg Leu Leu Glu Ala Ser Leu 530 535 540 Glu Glu LeuLeu Glu Val Glu Glu Val Gly Glu Leu Thr Ala Arg Ala 545 550 555 560 IleLeu Glu Thr Leu Lys Asp Pro Ala Phe Arg Asp Leu Val Arg Arg 565 570 575Leu Lys Glu Ala Gly Val Glu Met Glu Ala Lys Glu Lys Gly Gly Glu 580 585590 Ala Leu Lys Gly Leu Thr Phe Val Ile Thr Gly Glu Leu Ser Arg Pro 595600 605 Arg Glu Glu Val Lys Ala Leu Leu Arg Arg Leu Gly Ala Lys Val Thr610 615 620 Asp Ser Val Ser Arg Lys Thr Ser Tyr Leu Val Val Gly Glu AsnPro 625 630 635 640 Gly Ser Lys Leu Glu Lys Ala Arg Ala Leu Gly Val ProThr Leu Thr 645 650 655 Glu Glu Glu Leu Tyr Arg Leu Leu Glu Ala Arg ThrGly Lys Lys Ala 660 665 670 Glu Glu Leu Val 675

What is claimed:
 1. A thermostable DNA ligase, which is a mutant of awild-type thermostable DNA ligase having an amino acid sequence of SEQ.ID. NO: 97, wherein the mutant thermostable DNA ligase has a mutation inthe amino acid sequence of SEQ. ID. NO: 97 selected from the groupconsisting of a mutation changing amino acid residue 294 from lysine toanother amino acid residue, a mutation changing amino acid residue 415from cysteine to another amino acid residue, R337K, C412A, C428A, D120E,and D120N.
 2. The thermostable DNA ligase according to claim 1, whereinthe mutation in the amino acid sequence of SEQ. ID. NO: 97 is a mutationchanging amino acid residue 294 from lysine to another amino acidresidue.
 3. The thermostable DNA ligase according to claim 2, whereinthe mutation in the amino acid sequence of SEQ. ID. NO: 97 is selectedfrom the group consisting of K294R, K294Q, K294L, and K294P.
 4. Thethermostable DNA ligase according to claim 3, wherein the mutation inthe amino acid sequence of SEQ. ID. NO: 97 is K294R.
 5. The thermostableDNA ligase according to claim 3, wherein the mutation in the amino acidsequence of SEQ. ID. NO: 97 is K294Q.
 6. The thermostable DNA ligaseaccording to claim 3, wherein the mutation in the amino acid sequence ofSEQ. ID. NO: 97 is K294L.
 7. The thermostable DNA ligase according toclaim 3, wherein the mutation in the amino acid sequence of SEQ. ID. NO:97 is K294P.
 8. The thermostable DNA ligase according to claim 1,wherein the mutation in the amino acid sequence of SEQ. ID. NO: 97 isR337K.
 9. The thermostable DNA ligase according to claim 1, wherein themutation in the amino acid sequence of SEQ. ID. NO: 97 is C412A.
 10. Thethermostable DNA ligase according to claim 1, wherein the mutation inthe amino acid sequence of SEQ. ID. NO: 97 is a mutation changing aminoacid residue 415 from cysteine to another amino acid residue.
 11. Thethermostable DNA ligase according to claim 10, wherein the mutation inthe amino acid sequence of SEQ. ID. NO: 97 is selected from the groupconsisting of C415A, C415V, C415T, and C415M.
 12. The thermostable DNAligase according to claim 11, wherein the mutation in the amino acidsequence of SEQ. ID. NO: 97 is C415A.
 13. The thermostable DNA ligaseaccording to claim 11, wherein the mutation in the amino acid sequenceof SEQ. ID. NO: 97 is C415V.
 14. The thermostable DNA ligase accordingto claim 11, wherein the mutation in the amino acid sequence of SEQ. ID.NO: 97 is C415T.
 15. The thermostable DNA ligase according to claim 11,wherein the mutation in the amino acid sequence of SEQ. ID. NO: 97 isC415M.
 16. The thermostable DNA ligase according to claim 1, wherein themutation in the amino acid sequence of SEQ. ID. NO: 97 is C428A.
 17. Thethermostable DNA ligase according to claim 1, wherein the mutation inthe amino acid sequence of SEQ. ID. NO: 97 is D120E.
 18. Thethermostable DNA ligase according to claim 1, wherein the mutation inthe amino acid sequence of SEQ. ID. NO: 97 is D120N.